Genetics and Genomics Notes

8/10/2019 Genetics and Genomics Notes

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Genetics and Genomics

Forward geneticso Phenotype to genotype

Reverse geneticso Genotype to phenotype

Cell is the basic component of organismso Nucleus contains the geneso Mitochondria have their own genomeo Prokaryotic cells differ

Genetic material in a nucleoid region Cell is organized but has no organelles

Almost everything is encoded in the DNAo DNA karyotype-lay out chromosomes

Centromereo Helps the chromosomes migrate from the middle of cell to poleso Metacentric=middleo Submetacentric=below the middleo Telocentic=at the end

Cell division is essential to lifeo 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 chromatino Spindle attaches to kinetochore via the centromere

DNA replication can induce errorso Mutations or other changeso If it was perfect there would be no variation

Source for variationo DNA replication and repairo Crossing over and chromosome segregation

Cell cycle is monitored by checkpointso G, S, and Mo G0= nondividing cello Interphase is G and So The checkpoints can let mistakes through

They check for DNA damage or a failure to replicate Something is wrong=apoptosis

Phenotypeo Appearanceo What is expressed


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o Could be complex Genotype

o What do the genes sayo Homo/heterozygouso Dominant vs recessive

WT vs Mutanto Wildtype is the normal that is definedo Mutant is any changeso Only 1 WT, but many mutants

Mendelo Found that in the F1 generation only one gene/phenotype dominatedo But if F2 it was a 3:1 phenotypic ratioo Chose phenotypes coded for by 1 gene

Monohybrid crosso Only one gene being crossedo Start with homozygous parental strains

Recessive alleleso Only expressed when two copies of the gene are presento In most cases the WT is dominant, but WT can also be recessive

Homozygouso Two alleles the sameo Can be dominant or recessive

Heterozygouso Two alleles are differento

Dominant will be expressed in most cases Hemizygouso Only one allele present

Dihybrid crosso Two genes cross to see effecto 9:3:3:1 outcome in the F2 generationo Independent assortment

Independent assortmento Combine the probability of one trait w/ probability of getting anothero Multiply

Test crosso Can determine genotype if unknown but have a known phenotypeo Difference between homo and heterozygouso Cross unknown with homo recessive

If homo- get all dominant expression If hetero-get some recessive expression (1/2)

Human crosses


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o Multiple different disorderso Dominant diseaseso Recessive diseases

Pedigreeso Can follow a disease in a familyo Can determine its genotypeo Recessive-skips generationso Dominant- in all generationso 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 t he gene but dont 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 +Pbo P(a/b)=Pa/Pb

Binomial theoremo Used to calculate the probability of any specific set of pairs of outcomes among a large #

of potential eventso P=n!/s!t! X a sb t o S=# of a outcomeso T=# of b outcomes

Chi-square analysiso

Variation between the observed and expectedo 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 nullo Use a graph of degrees of freedom (# of phenotypes-1) and x squared to determine P

Classes of mutationso 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


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Dominant mutationso 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 dominanceo Homo/heterozygous express the same phenotype

Incomplete dominanceo Heterozygotes have an intermediate phenotype

Codominanceo Express both alleles in the heterozygoteo Ex: blood type

Recessive lethal mutationso The homozygous recessive is lethal and will not surviveo 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 locatedo If in the same place, the cross leads to a mutationo If in different places the cross leads to a normal phenotype

Sex linkedo Genes located on the x chromosomeo Males have to get their x from the mom and their y from the dad

Pedigreeso Again help see the expression pattern

Does a genotype always result in the same phenotype


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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

Functionso Replicationo Information storageo Info expression

Variation through mutationo Allows new characteristics to evolve

Central Dogma

o DNA Transcriptiono RNA

Translationo 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 doesnt mean more complexity o Such thing as alternative splicing

DNA as the genetic materialo Griffiths transformation

Found that transformation occurred by some moleculeo Avery, Macleod, McCarthy

Only when using DNAse did transformation not occuro 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 RNAo Reverse transcriptaseo Integration

Discovery of DNA


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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 To G is always bound to Co A + T + C + G = 1o G3Co A2To 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 backboneo 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 chargedo

If together-will repel each othero Need to make up the outsides, with the bases in the middleo DNA has a negative charge

Migrates to the Anode DNAs density

o G-C bond is more dense due to 3 H-Bondso The higher the G-C content the more dense the DNAo Different DNA melting points as a result

FISHo Test to detect Nucleic Acids

DNA replicationo Semi-conservative

Evidence=2 rounds of replication with labeled DNA strandso Many generations-only trace amounts of the old-mostly new

Bacterial Replicationo Starts at a single origin of replicationo Bidirectional replication


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DNA Polym READS from 3 to 5 and SYNTHESIZES from 5 to 3 a new DNA strand DNA polym

o I, II, IIIo All can proofread and replace their mistakes

Holoenzymeso Protein machine made up of multiple proteins and TFs

Bacteriao Origin of replication is defined by a repeated sequence (9 mer)o DNAa molecules bind and create an initial bubble of replicationo DNAb/c bind to the bubble and initiate helical unwindingo Primase adds an RNA primero DNA polym starts

Leading vs lagging strando All replication proceeds towards the replication forko One strand is continuouso One strand is discontinuous

Needs multiple primers Multiple okazaki fragments Ligase sticks together

DNA poly Io Replaces the RNA primer with DNA

DNA gyraseo Untangles the DNA helix

Speedo

Euk > Pro because there are multiple origin sites Euk are not circularo Have an issue with end of chromosomeso Some cells have telomerase, which acts as an end primer to avoid losing some of the

telomereo Most cells dont have telomerase and lose a sm all portion with each replicationo 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 placeo 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


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Transcriptiono Help get an RNA messageo Need a template strand of DNA to get to RNAo RNA is identical to the coding strand, but is matched up with the template strand

Prokaryotic cello Replication, transcription, translation occur in the nucleus (nucleoid region)o Need an RNA polymeraseo Scans for an RNA binding siteo Need the sigma subunit to recognize the specific initiation sequence

Nascent RNAo Transcript

Sigma factor dissociates after a few nucleotides of the RNA strand is built upo Only necessary for binding and recognizing the promotoro Recognize TATA box upstream

Operonso Genes often found in a segment togethero Get a polycistronic mRNAo Only found in prokaryotes

Ribosomes translate as mRNA is being transcribedo No posttranslation modificationo Occurs faster than in Euko Quickly ramp up protein production

Eukaryoteso Many more regulation of the mRNAo

Separated into compartments RNA typeso mRNAo tRNAo rRNAo miRNAo catalytic RNA

Chromatin in Euko Densely packed DNA and organized by histoneso Before transcription may need to modify the chromatin

Hetero/Euchromatino 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


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o Not a lot have it, but if a gene has it, it is essential to transcriptiono Binds the RNA polymerase after binding TATA Binding Proteino Allows for a transcription regulationo Brings other RNA poly to site to increase regulation

CAAT boxo Another example of a TATA like binding element

Enhancerso Specific sequence that can be located in front of, in, or after the geneo If located in the gene it keeps the gene from being translatedo Can activate or depress depending on location

TFo Generalized proteins that bind specific sequences to regulate genes and expression level

Transcripto Eukaryotes need to mature ito Add a methyl G cap and poly A tail to stabilizeo 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 geneso Genes also interact with each other in different ways (regulate)

Splicingo 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 intronand splices it out

o Group 2 mRNA needs snRNPs get a complex that forms lariat loops that splice out introns exons ligated

modify the transcript

o RNA editingo Substitution editing

Get a change of a nucleotide in a transcripto Two forms of a protein depending on editingo Insertion/deletion editing

Can alter the function and shape of protein Or bring proteins into the proper reading frame to establish function


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Translation

The codon table Code for an AA Start codon AUG begins the open reading frame

Errors:o Spontaneous mutations lead to base pair changeso Point mutations: changes a proteino 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 tRNAs with AAs o Need an empty tRNAo AA synthetase puts the AA on the tRNAo Need ATP energyo Activated enzyme complex (AA, AMP, aminoacyl tRNA) attaches the AA to the tRNA

Factors associated with 3 different phases of translationo Initiationo Elongationo Termination

Ribosome is not formed until the mRNA binds the small subunito Then the large subunit binds

3 ribosomal siteso Aminoacyl site=AA sits in the tRNAo Peptide=growing peptide chaino Exit

Stop codon causes the complex to fall aparto Releases the peptide

Multiple translational complexes form on a single mRNA


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Amino Acidso R Group only thing that changeso Hydrophilic/hydrophobico Polar (charged)

The r group differs in forl/functiono Change the folding by mutations

Protein sequenceo Primary=AA sequenceo Secondary=alpha helix or beta sheet (H-Bond stabilized)o Tertiary=whole protein foldingo 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 modifiedo Add carbs to the proteino Golgi editing

Functions of proteinso Structuralo Contractileo Signalingo Storageo Transporto Enzymatic

Roleso

Enzymatic Lower activation barriero Signal sequence-domain that attracts substrateo Membrane anchoring

Mutations

Germline vs somao 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


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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 mutationo Deamination

Amino group in C or A converted to a keto group, which changes the basepairingo Depurination

Lose a nucleotide within the DNAo Oxidative damage

Oxygen damages the DNAo Transposons

Pieces of DNA that can insert or move within the genomeo 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 genotoxicityo Before anything is released used the Ames Testo Have a control side and get the number of random background mutationso Add the mutagen, see if any difference than the background rate

Repair

o DNA polymerase can proofreado 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


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REC-A comes back and fills in the gap DNA ligase ligates it together

o SOS repair Last resort Induces more mutations Only occurs when there is massive mutation

o Photoreactivation repair Dimer forms Dimer repaired Normal pairing restored

o Base excision repair Recognizes a single wrong nucleotide Base removed by DNA glycosolase AP endonuclease recognizes lesion and nicks DNA DNA polymerase fills gap

o 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 evolutiono Species have a common ancestor

Neodarwinism

o Discovery of genetics Evolution requires:o Variation between organismso Competition between individualso Selection

Descent from common ancestorso Can use genetics to find these relationships

Two formso Micro/macroevolutiono Large and small scale

Phylogenetic tree-shows relationship between specieso Stasic- doesnt change o Anagenesis-one species evolved into a different oneo Cladogenesis-species diverged into 2 separate ones

Morphologyo Species based on the way they look?o Not a great model due to different looking organisms being of the same species


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Biological species concepto Define a species based on the ability to reproduce and have offspring

Selection and fitnesso Advantage for one characteristico Get some fitness affecto Fitness-measure in the success of breeding

Mutations usually arent good o However may be advantageouso Selected for

Stabilizing selectiono Less genetic variationo When the environment is stable

Directional selectiono Shift towards one sideo When the environment changes

Disruptive selectiono Environment heterogeneouso Can harbor two different organisms

Maintain genetic variationo Variation is not limitedo Sequence the genome to see the differenceso Change environment, some mutations become advantageous and are selected for

Cost of variationo Protective effects of sickle cell anemia against malariao

Fitness to genotype changes with the environment Speciationo Pre/postzygotic barrierso Ex: geographical separation

Population geneticso Hardy Weinberg

Describes an ideal populations allele and genotype frequencies P2+2pg+q 2=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 environmentchanges

Genetic drift


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o The changes in allele frequencies due to chanceo More of an effect in a smaller population

Founder effecto When a new population is started due to migrationo Will not have the same allele frequencies as before

Inbreedingo Inbreeding depression (lose heterozygotes)o No new influx of genetic materialo 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 moleculeso Bacteriophage=lambda (lollipop head)

Circular replicationo Cut bu a nucleaseo Copied discontinuously and continuously

Bacterial DNA packagingo Ecoli supercoils the DNAo DNA has no tension due to turns

Eukaryoteso Organize using histone proteinso 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 DNAo Centromeres

Sister chromatid cohesion Assembly site for kinetochore


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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

Epigeneticso Histone modificationo Can be passed ono Reversible

Epigenatorso Environmental signals (internal or external)o Signal is transduced to the cell

Histone modificationo Histones have a tail that can be modified

Acetylationo Opens upo Deacetylation closes

Methylationo Opens or closes depending on location

HDACo Histone deacetylation complexo

Closes the DNA up HATo Histone acetylation complexo Opens dna up

CPG islandso Sites where the DNA is methylated

Imprinting


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o IGF2 not turned offo Hypo/hyper methylationo Epigenetic inheritance can lead to cancer

Variation in chromosome number and arrangement

Karyotypeo Group chromosomes and banding patterns

Aneuploidyo 2nx chromosomes

Euploidyo Multiples of n chromosomes

Polyploidyo Multiples of the same geneo Auto/allopolyploidy

Auto=duplication of whole genome Allo=duplication of 2 diff species

Nondisjunctiono Doesnt separate o Leads to trisomyo Trisomy 21=downso Trisomy 13=patauo Trisomy 18=Edwards

Chromosomal rearrangementso Need breakage of a chromosomeo Terminal deletion (lose piece at origin)o Intecalary deletion

Form a deletion look and it ejects a gene out of a chromosomeo Deletion loop

Duplicationo Unequal crossover between 2 sets of homologous chromosomeso rRNA present in many copies

CNV (copy # variants)o Chunks of repeated DNA in chromosomes due to duplicationo Can be present within promotor regionso Can cause an increase in the replication of the gene

Inversiono May express new geneso Can happen due to loopo Forms a 4 part breakage

Paracentrico Doesnt change arm length


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Pericentrico Changes the length of the arms

Consequences during chromosomal inversiono 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 chromosomeso Forms a cruciform tetrad during meiosis

Robertsonian translocationo Exchange of small arm of one chromosome for the large arm of anothero Can get familial downs

Fragile Xo Pieces of the X can break off at the endo Its so thin because the DNA isnt 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 experimentso Just the medium allowed to pass

Conjugationo Needs a sex pilus and attachment between cells to pass the DNAo Also need a plasmid F factoro Will not happen in U tube

HFR cellso Have the F gene in the DNA itselfo Will conjugate but will not pass on the F gene to the other cell


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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 its the HFR cell Transformation

o Take up free genomic DNA from the environment and incorporate ito Will occur in U tube experiments

Transductiono The DNA is inserted via bacteriophage into another cello Will occur in U tube experiments

Genetic mappingo Use recombination between the regions to map for mutationso Deletion mapping-map consequenceso Recombination mapping-based on genetic exchange

Linkageo Two genes on a single pair of homologso No exchange occurs

Distance matters in recombinationo Count the recombinants and parentalo Map distance=REC/(total) X 100o 1cm= 1% recombination observed

Two and three point mappingo Consider single and double crossovers

Double cross overso Frequency is the product of the two SCOs

Tableo The highest #s are the parental strainso The lowest numbers are the double crossovers

Order of the genes is based off of which one is in the middleo For the double crossover, the one that appears to change is the middle

C=coefficient of coincidenceo DCO observed/expectedo Interference=1-C

Never the expected due to one crossover inhibiting a second Somatic cell hybridization

o Linkage mapping Sister chromatid exchanges

o Dont see phenotype exchanges


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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)

Chloroplastso Larger DNA than mitochondria (more introns)

MtDNAo Smallero Goes missingo No introns

The origin of mitochondria is via the endosymbiosis theory Nuclear contributions to the mitochondria and chloroplasts

o Via nuclear geneso Passed on via regular genetics

Mitochondrial diseaseso MERRF, LHON, KSS

Genetic elements + viruses

IS elements (bacteria)o Insertion sequenceo Defined by inverted terminal repeatso Flanked on both sides of the geneo 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

Heteroduplexo The complementary sequence that helps it bud off

In the presence of Ac, Ds is not transposableo But Ac alone can transpose


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o Ac must still have its transposase geneo 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 transposonso Get crossovers between repeatso Get circular deletiono Separate from chromosome

RNA based TEs o Retrovirus

LTR=attracts RNA polymerase to make its productso LINEo SINE

Replication (copy elements)o Transcribed into RNA and proteino Can silence the transposon DNAo Target for destruction

Retroviruso Integrase

Mediates integration of DNA into genomeo Retroviral integration

ssRNA to dsDNA reverse transcriptase cant proofread

o retroviral budding products packaged and moved to the PM

DNA viruseso Have a lytic/lysogenic life cycle

RNA viruso Remain RNA alwayso 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


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Select cells with recombinant DNA by antibiotic resistance selection Libraries

o Collections of cloneso CDNA library (complementary DNA)o Higher complexity means the more coverageo 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 DNAo Phage/cosmid vectors are largero Artificial chromosomeso Expression vectorso Shuttle vectors

Restriction Enzymeso Endonucleases with a specific recognition restriction site where it cuts DNAo Leaves sticky endso Cut every 4 N 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=denatureo

50=annealingo 75=polymerizationo Amplify the DNA experimentally

Real time PCRo Probe on the templateo See the florescence level

Restriction mappingo Cut the DNA with different enzymes and see how the DNA is put together

Southern Blot=DNA Northern Blot= RNA

Genomics

Sanger sequencingo Able to do short segments of the genome (about 1000)

Next gen sequencingo Sequences the entire genome

Clone by clone sequencing


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o Cut up the genome into pieces using REs o Smaller and smaller pieceso Then insert into a large plamid

YAC/BACo Fit together with overlapping clones

Shotgun based sequencingo Use different REs 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 frameso Best when there are no introns

Databaseso BLAST

Uses an algorithm to see how close a protein overlaps with the alignment ofknown proteins

Determine % overlap Also can determine functional domains

Human Genome Project (HGP)o

20,000 geneso 3 billion base pairso 98% noncodingo The noncoding DNA may have a regulatory functiono Made partial chromosomal maps

Genes clustero Deserts in between genes

Disease mapso Map the genes that cause diseases and where it is located on the chromosome

ENCODEo Look at hetero/euchromatin and changes from cell/cello Shows where the genes are going to be expressed

CHIPo Chromatin immunoprecipitateo tag the protein with antibodies to find the protein of interest

Omics


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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 SNPs Single mutations at a specific location

o CNV Copy number variants Large piece of DNA repeated

GWAS databaseo Links phenotypes to genotypeso Associates SNPs with genomes

Need to adjust the p value when you have such a large sample sizeo Bonferroni-corrected significance cutoff

Original p / N (sample size) Pharmacogenomics

o Try to associate peoples genomes to the way a drug functionso 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 persons genome

Adverse drug reactionso Cost billions of dollarso People process drugs in different ways

Ultrarapid metabolizer > extensive metabolizer (normal) > Poor metabolizers

Prokaryotic gene regulation

Operonso 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 necessaryo System can be turned on/off depending on environmental stimulio Positive control


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Inducer in the system that turns on gene expressiono 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 clusterso 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 transcriptiono 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 constantlyo LacI mutants

Can bind to the promoter but not lac, so always offo Operator region

Wont bind the repressor-always on Known as the O c 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 effecto Repressor can be made anywhere and travel to bind the promotero IPTG can induce the lac operon expreeion

Glucose is the preferred carbon sourceo Less energy cost to the cello Glucose levels high, cAMP levels low

cAMP levels are high when no glucose


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o cAMP binds to CAP (catobolite activating protein) CAP induces expression of the lac operon (assuming lac is present)

Lac repressoro Homotetramero Inserts 4 O sequences that then are pulled together to form a repression loop and stop

transcription Trp operon

o Repressible systemo Opposite of laco The presence of trp shuts off the operono The lack of trp turns it ono The repressor is bound to the operon when it is bound to trp

Trp mutantso trpR mutants

always ono trpO mutants

always on because it cant be blockedo trpP mutants

always off attenuation

o an interaction between transcription and translation that regulates expressiono 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 tRNAs 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.colio Both types of control are mediated by Ara C

Dont invest in the synthesis of any other sugar if glucose is present

Eukaryotic gene expression

Domains separate chromosomeso Chromosome territories


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o Interchromosomal domains between chromosomes Compactness of DNA

o Acetylation and methylation of histones and DNA regulate the compactness ofchromatin

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 transcriptiono Insulator elements prevent the spread of chromatin remodeling

Cis acting sites in chromosomal DNA bind to transcriptional regulatory proteinso Promoterso Enhancerso Silencers

Promoterso Focused

Always initiates transcription from the same siteo 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 TFs o Effect of mutations

Mutate promoter elements-reduce the transcription level Cis acting elements bind TFs o TFs often expressed in time and tissue specific patterns and can recruit or interact with

RNA polymera se, and other Tfs, and respressor proteins Basal transcription level vs induced transcription level Functional domains of TFs

o Can screen the genome and ID the TFs based on their properties


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o DNA binding domains Helix turn helix Trans-activated domains (repressors) Zinc finger DNA binding domain Basic leucine zipper

Assembly of TFso Ex: RNA polymeraseo TBP (Tata Binding Protein)

Binds to the sequence and brings in TAF (TATA associated factors)o Polymerase comes in and forms the complexo TBP and TAF stays in place to recruit additional transcription complexes

Enhancerso More upstreamo Help attract TFs o Can affect how fast a complex is madeo Increase the rate of DNA unwinding and RNA polymerase release from the promoter to

initiate transcriptiono Ex: UASg

Constitutively active post translational regulation

o alternative splicingo 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 levelo 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 inthe 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


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both bind to the RICS and RITS complexes created from dsRNA via the dicer protein

o RISC Degradation of mRNA complementary to the sequence of the small RNA Downregulates the mRNA that is not exactly complementary but close

o RITS Goes directly into the cell nucleus and downregulates the production of the

gene directly

Gene Function

Forward geneticso Genome wide genetic screens for mutants with specific phenotypeso Id the genotype that creates the phenotype

Reverse geneticso

Define every gene in the genome based on sequence analyseso Reduce/eliminate functions of specific genes and assess the phenotypic impacts

Model organismso Easy to growo Short generationo Abundant progenyo Can cross in large numbers

Yeasto Simplest eukaryoteo Haploid and diploid alternating generations

o Phenotypes are evident in haploido Diploid allows for recessive lethal mutations to be studied

Drosophilao No meiotic crossing over in maleso Diploido Recessive lethal mutations are maintained in strains heterozygous for balancer

chromosomes P-Elements

o DNA transposons that insert into the genomeo 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 geneso Need a positive selective markero Can use this to destroy genes

Randomly inserts itself into the open reading frameo P elements either insert or destroy gene


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Miceo Genomic synteny with humanso Large scale genomic screens difficulto Creating transgenics and gene knockouts/replacements is more feasible

Mutagenizationo 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 mutationso Genetic screen helps select out the ones which were mutatedo 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 stresso Screening mutants (Balancer Chromosomes)

Can screen for recessive mutations in diploids by creating a collection ofmutagenized chromosomes in balanced heterozygotes

Assess the phenotypic impact of homo/hemizygocity Retain mutations of interest

Untangling pathso The order of generation is based on epistasiso The effect of mutation in one gene masks or modifies the mutation in another geneo Pathways affected by this-because every gene must be present to make a producto Use epistasis analysis to determine which gene is at the top and which is at the bottom

Can determine pathway order with mutation to geneso Screens for suppressor mutations can ID additional genes in a pathway not Ided 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 phenotypeo Suppressor mutants

Diminish/eliminate the phenotype caused by the initial mutation Gene product sequence

o May reveal gene functiono Presence of domains known to have specific functions


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Gene product functiono Investigated further using molecular genetic tools and techniqueso 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 libraryo Use antibodies against the protein of interest to screen expression vectors/libraries

Expressed clone contains sequences for the gene of interesto 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 expressiono Want to ID where and what the gene is doing in the cellso Place and time of gene expressiono Tagged immunochemistry/florescence to see where and when the protein is expressed

Miceo Selection for insertion of positive selectable marker disrupting the target geneo Get recombinant and negative selection against nonhomologous insertionso 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 interferenceo 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. Productso cell lines genetically engineered to produce a medicine/drug

biologics produced using bacteria, fungi, and cell lines as bioreactorso Can create insulin for exampleo Extract the A and B proteins from different cells and combine them to form fully

functional insulin Biopharming


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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 viruso Can be edible or injected

Subunit vaccineo One or more surface proteins from a pathogeno Get an immune response

Inject proteins into a person-still get the immune response Plant genetic engineering

o Higher yieldso Drought preventiono Started from artificial selection

Select the best ones and breed them EPSP

o Important to produce aeromatic DNAo We dont make some of these AAs

Tyrosine, threonineo Bacteria and plants make themo Destroy operation of aeromatic AAs 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 thingso Constitutively on promoter turns on when the object is present

Synthetic bioo What is the minimum genomeo Can then begin to incorporate other things

Fetal karyotyping and genotypingo Amniocentesis

Stick a needle in and take amniotic cellso Chorionic villus sampling

Take a sample from the placentao Fetal cell sorting

Blood sample from mother (some fetal cells)o Helps get a karyotype and genotype on fetal cellso Preimplantation diagnosis

PCR amplifying DNA RFLP


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o Detect 5-10% of genome wide sequence variation Aso testing (allele specific oligonucleotide)

o Short oligonucleotide of defined sequence based on SNPs hybridization PCRamplification of genomic DNA from sample

Array based genotypingo Microarrayo Look for gene expression and levelo check for SNPs/CNV variation

p53 genechipo 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 fixo never officially proven

MMLV virus used to insert the correct geneo Virus that effects once and shuts down

Majority of delivery vesicles are viruseso Randomly integrate-so need better control

Concernso Capacity only 8kbo Could provoke an immune response

Body Plan

Developmental geneticso Genetic and molecular mechanisms underlying cellular and organismal development,

homeostasis, aging and senescence Development

o Develop tissueso Death of specific tissueso Balance between growth and death

Specificationo When genetic and positional cues confer a spatially discrete ID on cells

Determinationo Cells time when a specific developmental state becomes fixed

Differentiationo Process by which a cell achieves its final form and function

Hypothesiso Development-attainment of a different state by all somatic cells in an organism

Variable gene activity hypothesiso Differential expression and action of genes


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Controls developmento When and where are genes expressed and activeo How is gene expression regulated

Preformationo Sperm had little human inside that became bigger

Fertilization occurs when an egg and sperm fuseo Maternal cytoplasmic componentso mRNA and proteins

first components to trigger development without these nothing would happen

body plano very similar in organisms within the same specieso Pattern of organization-characteristics and recognizable traits

Pattern formationo Aspects of development of the body plano Leads to genesis of patterns or structures that make up the body plan

Number of axes (primary)o Anterioro Posterioro Dorsalo Ventral

Animal body plans are segmentedo The body plan has 11 segmentso Often has appendages

Drosophila

o Homologies among embryonic, larval, and adult body planso Governed by a set of geneso 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 cellularizationo The pole cells form at the posterior and are the precursors to a germ cell lineo Maternal functions direct the AP and DV axes

Zygotic geneso Part of the genome but regulated by maternal effect geneso Gap genes, pair rule genes, and segmental polarity genes form the body plan


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o Then homeotic genes (HOX genes) determine the fate of cells and specify the type ofcell they will become

Nuslein-Volhard and Wieschauso Determined which genes are important to body plan

Maternal effect geneso Form the anterior posterior gradiantso Gap genes are triggered (they are TFs)

Trigger certain genes in gap genes to form the band regionso Formation of discrete bands triggers pair rule genes

Divide gap gene bands into smaller regionso Activation of pair rule genes activate segment polarity genes

Even more dividedo Then the hox genes are activated and specify the ID of each segment

Gap genes are zinc finger TFs o Activate the next set of genes

Pair rule geneso Often encode helix turn helix TFs o Overlap of TFs or non overlap specifies specific segments

Mutationso Runt (mutated RunX2 protein) encodes a TFo Mouse doesnt have proper muscle/bone development o Humans get cleidocranial dysplasiao Autosomal dominant diseases

Two Hox genes clusters in drosophilao

Antennapedia complex and Bithorax complex Hox genes and TFs with Homeobox o DNA binding homeodomaino 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 DNAo Not intermixedo Collinear with expression patterns in the embryo

Humanso 4 human hox gene clusters 39 totalo Control A-P patterning in humans (and other vertebrates)o 5 end geneso Limb developmento Humans dont get mutated very often (need a double mutation since diploid)

get smaller changes (ex: polydactyly)


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o occurs in c elegans to get to the 959 cells hermaphrodites 131/1090 cells die males 147/1178 cells die

o 15 cell death (ced) geneso Apoptosis=cell death (programmed)o Ced9 expressed, shuts down ced3-4 and the cell survives

Vice versa, no ced 9, get cell deatho Gain of function mutation in ced9- leads to no cell deatho BCL-2 is the human version of ced9

Overexpression prevents cell death

Cancer genomics

Disease of somatic cellso 25-33% of the human population is affectedo 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 growtho Metastasis-movement of cancer cellso Benign tumor- local mass of cellso Malignant tumor-cells metastasize and the tumor has access to a blood supplyo Primary tumor-the initial site

o Secondary tumor-the site where the tumor spreads to Genetic theory of cancero Cancer is the result of multiple gene mutationso Accumulation of mutations in different genes due to genetic or epigenetic variationo Up to 10 10 mutations over a human lifetime

Genomic instabilityo Mutator phenotypeo Aneuploidyo Rearrangement

Translocation

Inversion Deletion

o SNPs o Amplification

Clonalityo Tumors are comprised of clonal cell populations that all originate from a single founder

cell


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o Disregulated growth and then disregulated movemento 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 growtho 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 controlo Shut down cell division if activatedo 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 5o Keep growing- dont stop division o APC=tumor suppressor geneo Role in contact mediated growth inhibitiono Get polyps in the SI

Driver mutationso Confer growth advantage to cancer cellso Cancer becomes worse with these mutations

Passenger mutationso Other mutations that happen in the course of cell division that do not confer growth

Epigenetic variationo

Demethylation or acetylation of chromatin encompassing genes that stimulate celldivision/migrationo 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 celldivision and excessive cell proliferation

o Abundance of different cyclins during the cell cycle that regulate transitions from onepart to the next

o Mutate cyclins

Get cell d ivision when cell shouldnt be dividing Apoptosiso Programmed cell deatho BCL 2 level important (low for cell death)o Cell death triggered by caspaseso Apoptotic bodies are engulfed by phagocytosis

Bax homodimer promotes apoptosis


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o P53 induces BAX transcription Inhibits BCL2 transcription This stimulated cell death

o P53 low function in cancer BAX transcription low BCL2 high Cell doesnt die

RAS (protoonco gene)o GF stimulates the cell proliferationso Activated when bound to GTPo Tells the cell to proliferateo Mutated and constantly active-cell always proliferates

P53 (tumor suppressor)o DNA damage repairo DNA damage promotes cell cycle arrest and fixing of the DNAo Mutation rate increases if p53 not working

RB1 (tumor suppressor)o Inhibits TFs when not phosphorylatedo Can be inherited- one copy damagedo Triggers a cascade of genes that pushes the cell through the cell cycleo No longer binding E2F?

Constantly pushes the cell through the cell cycleo Only one good allele needed

Migration of metastatic cells away from the primary tumor siteo Establishes itself at secondary tumor siteo Get blood vessels to oxygenate (angiogenesis)

Metastatic cellso Reduced expression of E-cadherin glycoprotein (reduced cell-cell adhesion)o Increased expression of tissue metalloproteinases (TMPs) (increase cell migration)o Reduced interaction with tissue inhibitors of TMPs (increase cell migration) o LOF mutation in metastatic genes-leads to metastasiso Or GOF mutation

Viral contributions to cancero Many people believe that cancer is caused by a viruso

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


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o Radiationo Smokingo Other factors

Drug designo Use the exact path to develop drugso Many times specific to a certain mutationo Gleevec

Acts as ATP and binds the site that allows BCR-ABL to stimulate cell divisionwhen 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

Genetics and Genomics Notes

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