Lecture 1 Notes Basic Definitions

- Allele: alternative forms of a gene that arise by mutation and are found at the same place on a chromosome

- Genotype: all genes in your genetic code - Phenotype: way your genotype is presented physically, your presented characters - Penetrant: proportion of population that has a certain genotype AND displays that phenotype - Expressed: the level at which the gene manifests from individual to individual - Epigenetic: other factors that change expression of genes (ex. via change in regulation like

methylation) Genetic Determinism

- Soft Determinism: genes affect healthy/diseased states but act along with environmental factors, history, epigenetic modification

o Multifactorial aspect: healthy and diseased genome could look the same - Strong Determinism: genetics PRIMARY drivers of health and disease; other effects are

MODIFIERS (e.g. diseased and healthy genomes would look v. different) o Alleles will be highly PENETRANT and EXPRESSED and have high value as PREDICTIVE

BIOMARKERS OF DISEASE - Problem: probably varies by disease/condition

DNA Makeup - Nucleotides: “parts” of DNA (AGCT, T U in RNA) - Single strand has a linear pattern of reading 5’ 3’ - 2 COMPLEMENTARY strands per molecule of DNA that fit together ANTI-PARALLEL via H-

bonding Building a Molecule of DNA

- Oxy sugar: 2’C has an OH found in RNA (left pic) - Deoxy sugar: 2’C has an H found in DNA (left pic, right side)

o P is covalently bound to 5’C (if 3 - α, β, γ) o Nitrogenous Base is covalently bound to 1’C o Next nucleotide attaches onto the 3’C

o Monophosphate: free in cell o Synthesis—adding to the 3’ end—cleaves off β and γ-phosphates as inorganic P o α-Phosphate is incorporated into phosphodiester bond o need TRI-phosphates for formation of phosphodiester bonds, cannot use mono/di

Purines and Pyrimidines - Purines: Adenine, Guanine

o RNA – Adenosine, Guanosine DNA – Deoxyadenosine, Deoxyguanosine

- Pyrimidines: Cytosine, Thymine, Uracil o If find uracil in DNA, it is due to a postpolymerization chemical change in structure

Base Pairing - T-A = 2 hydrogen bonds (“TATTOO”) - C-G = 3 hydrogen bonds takes more E to disrupt

Phosphodiester Bond - Linkage between the free –OH group of the 3’C and the phosphate group on the 5’C - DNA polymer has 5’ to 3’ directionality - Synthesis always adds onto the 3’ end - PHOSPHATES and SUGARS form the PHOSPHODIESTER BACKBONE via phosphodiester bonds - Phosphate backbones do NOT interact w/ each other b/c they are (-) and repel - NITROGENOUS BASES do interact w/ each other via H-bonding

Lecture 2 Notes General Notes

- Sequence of nucleotides determines complementarity on other strand - 5’ CGT 3’, complementary strand 5’ ACG 3’ (antiparallel) - H is covalently bound to electronegative elements interacts w/ other electronegative elements

(H-bond) - TA = 2 H-bonds - GC = 3 H-bonds - Chargaff: noted 4 bases found in characteristic ratios (A=T, G=C)

General Structure of DNA Double Helix - 10 bases and 34A (3.4nm) = 1 complete turn; helix = 2nm wide - Antiparallel strands - Right-handed helix - 2 asymmetrical grooves: major + minor

o Major = where most base pairs project (0 charge, where proteins usually interact) o Minor = mostly phosphodiester backbone (- charge, makes it hard to access interactions

+ small) - 5’ end = , 3’ end = - Only 25% genome is genic; exons = 1% of genome

DNA Replication - Base-pairing: lets DNA (nucleotides) serve as templates for new complementary strands - DNA replication is MUTAGENIC b/c DNA polymerase makes errors

Semi-Conservative DNA Replication - DNA unzips and newly synthesized strands form complementary to each strand of DNA - Leading strand: 3’ end toward fork, allowing synthesis to be continuous w/ that strand (DNA

builds 5’ -> 3’ and adds onto 3’ end) - Lagging strand: 5’ end toward fork, requires Okazaki fragments

Packaging the Helix 1. DNA (2nm) is wrapped around histone protein cores 2. 8 histones make an octamer called a nucleosome (10nm) 3. Nucleosome fibers associate to form solenoids (30nm) 4. Solenoids loop and wind to form interphase chromosome - Makes DNA more resistant to shear forces

Studying Cell Cycle in Humans: Colchicine + Cytochalasin D - Colchicine: microtubule spindle poison, WBCs + colchicine = stop @ metaphase, no spindle

apparatus (should see fully condensed, replicated chromosomes, gives good visual of all your chromosomes)

- Cytochalasin D: actin poison, stop@ interphase (should see loosely wound DNA, hasn’t been replicated)

Chromosomal Typing - Chromosomes at different stages of cell cycle show diff. levels of condensation - Interphase: least condensed, DNA associated with nucleosomes, but not wound further

o Loose = good for replicating, transcribing (G1, G2) - Metaphase: chromosomes tightly condensed - Metaphase to Anaphase: most condensed

Visualizing Chromosomes - Giemsa Staining: gives G-banding of chromos. isolated from mitotic metaphase arrested cells,

contains lots of methylene blue

- Methylene blue (+): will bind less condensed DNA, esp (-) phosphodiester backbone - When using mitotic metaphase chromos, single chromo has been through S-phase and

replicated, so every chromosome has 2 chromatids (not yet chromatid chromosome); therefore 2 sister chromatids are staining controls for each other, because should see same bands on each one

Visualizing Chromosomes: Centromere, Kinetochore, Telomere, Structure - Centromere: constricted center of chromosome, links sister chromatids, region IS replicated, - Kinetochore proteins: protein structure on chromatids where spindle fibers attach and pull

chromatids apart - Telomere: ends of chromosomes - Each chromatid is a DNA helix - 4 names for structure of chromosome:

o Metacentric: centromere in middle o Submetacentric: centromere closer to one end o Acrocentric: centromere EVEN closer to one end o Telocentric: no/small p arm (essentially just 1 arm)

Visualizing Chromosomes: Cartoon Karyotype - Early = more solvent available for methylene blue to bind b/c less tightly wound (more bands!) - Late = bands fuse together, regions become inaccessible as tighter wound (fewer bands!) - Banding pattern, size, shape help define which chromosome you’re looking at - Normally, L/R sides are matched—this cartoon karyotype is just visual

Human Karyotype: Chromosomes from a Metaphase Cell, Geimsa Staining and G-Banding - Karyotype at metaphase-arrested point in cell cycle - 22 autosome pairs (1 copy from mom, 1 copy from dad = 44 total autosomes) - 2 sex chromosomes = 46 human chromosomes - Complete androgeny insensitivity syndrome: XY appears normal but person presents as

completely F, sterile - XX males: part of Y has been translocated onto X

Makeup of the Human Genome - 1.5% Exons: protein, rRNA, tRNA coding region - 24% Introns, regulatory sequences: gene-associated sequences, promoters - 15% Repetitive DNA unrelated to transposable elements, large-segment duplications - 3% Simple sequences DNA - 10% Alu elements: reverse transcribe small RNAs - 44% Repetitive DNA including transposable elements and related sequences - 15% Unique noncoding DNA (???) - Non-exon and intron portion: “dispensable” portions could be here and you would never be

able to tell phenotypically (could be missing 1 mill base pairs!) - LOTS of noncoding portions in the genome

o THEREFORE no 1:1 correlation b/w gene density and chromosome size Cell Division Review

- Mitosis: 2n mother produces 2 x 2n daughters (double amt. genetic material) - Meiosis: 2n mother produces 4 x 1n daughters (reduce # chromosomes in half)

Mitotic Cell Division Cycle - 2 major phases: M Phase ([P, M, A, T], C) – everything moving around, Interphase (G1, S, G2) –

everything replicating and getting ready - G1 10 hours, cell grows and carries out normal metabolism, organelles replicate - G0 (option) quiescent stage cells that have exited G1 and M phase

- S DNA replication and chromosome duplication - G2 Cell grows and preps for mitosis - Sign of oncogenesis G0 cell moves to G1 - Transitions b/w phases are regulated by mitotic checkpoints - Mitosis = cell replication because:

o 2 daughter cells are produced from 1 progenitor, nucleus and cytoplasm divide o Each daughter cell carries identical copies of the progenitor chromosomes, so each

daughter has IDENTICAL genetic info in the nucleus as the progenitor

Interphase - Chromosomes not highly condensed - Ploidy: number of copies of each chromosome - Diploid: 2 copies (homologs) of each chromosome - Haploid: 1 copy of each chromosome - Diploid number = 2n (in example) if replication, still 2n

- C number = 2c (in example) if replication, becomes 4c - Diploid number: - C number: # of copies of each chromosome

Metaphase - Chromosomes condensed and lined up on metaphase plate - Cell still DIPLOID, b/c sister chromatids have not yet separated - 2n, 4c

Anaphase - Kinetochores separate, spindle microtubules pull chromosomes away from each other twd

centrioles; 2 daughter cells still share same cytoplasm - 2 chromatids that made up 1 chromosome now each become a separate chromosome (“tid to

some” transition) - Karyokinesis: dynamic assortment of chromosomes

Telophase - Spindle breaks down and nuclear membrane reforms

Cytokinesis - 2 new daughter cells, each identical in genotype to mother cell

Lecture 3 Notes Meiosis

- Reductional division, 2n 1n - After meiosis 1, daughter cell is 1n, 2c (chromosomes haven’t yet separated) - After meiosis 2, daughter cells are 1n, 1c (chromosomes separate)

Post Meiotic Differentiation - Isogamy: same gamete forms in both sexes - Heterogamy: different gamete cell type in each sex (meiosis distinct, too!)

Summary of Chromosome Mechanics During Meiosis 1. DNA Replicates, Kinetochores Do Not 2N but 4C 2. Homologs Separate During Meiosis 1 1N but 2C 3. Centromeres Replicate, Separate = Meiosis 2 1N and 1C 4. Gametes Differentiate, Fertilization!

Meiosis by Mom: Big Investment, Expensive Gametes - Female ripens 1-3 follicles per menstrual cycle that then produce 1-3 oocytes 1. In F, many germ cells arrested during embryogenesis (dictyotene arrest) at meiotic prophase 2. Puberty, 1-3 germ cells transition to 1° oocytes 3. These oocytes complete meiosis 1 first polar body (1n) + secondary oocyte (1n)

a. Polar body = mostly nucleus, not much cytoplasm, divides to produce 2 more polar bodies (1n)

b. Secondary oocyte polar body (1n) + egg (1n) - 3 polar bodies never used in fertilization and chill under zona pellucida, may help define axes of

embryo Meiosis by Dad: Sperm are Cheap

- 280 mill. sperm/ejaculate 1. Mitotic proliferation of spermatogonia 2. Spermatogonial stem cell transitions to 1° spermatocyte and goes into meiotic cell division 3. Complete meiosis 1 + meiosis 2, karyokinesis but not cytokinesis (cytoplasmic bridges connect

spermatids) 4. Nuclei migrate into cytoplasmic vesicles (future sperm body) 5. Sperm breaks cytoplasmic bridge and swims off

Chromosomal Mechanics: Mendel - Gametic genotypes are driven by cell division - Monohybrid cross: het x het cross for alleles of ONE gene (ex. Dd x Dd for tall/dwarf)

Chromosomal Mechanics: Meiosis - If you start w/ heterozygote, as you go into Meiosis I, will replicate chromosomes and every

homolog has 2 copies of a chromatid - Recombination DOES NOT change gametic frequencies b/c 2 chromatids are identical - If NO recombination = alleles segregate in meiosis 1 - If YES recombination = alleles segregate in meiosis 2 - Preprophase: everything gets put onto spindle apparatus - Anaphase I: chromosomes segregate away, reductional division (2n=2, 4c 1n=1, 2c)

Mendel’s First Law - Mendel’s First Law: Genes are discrete entities that do not lose their identity during sexual

transmission from parent to offspring (not like mixing paint! If you are Dd, the d is still there but recessive.)

- Dominant alleles can conceal the presence of a recessive allele in the phenotype of a heterozygote

- In a heterozygote, 2 diff. alleles of a gene segregate away from each other during meiosis o Meiosis 1 if recombination o Meiosis 2 if no recombination

- Gametes are mutually exclusive! (D OR d, but cannot have a gamete that is Dd) - Gametes are produced by mutually exclusive events that generate their genotypic frequencies! - Fusion of gametes represents a statistically independent event BUT each different fusion

pattern represents a mutually exclusive statistical event - Independent gamete fusion: D AND D, or D AND d, or d AND d

Probabilities - For 2+ mutually exclusive events, ADDITION RULE - Independent events, PRODUCT RULE

Common Human Macromutations - Aneuploidy: due to meiotic catastrophe, centromere isn’t appropriately pulled around during

anaphase, chromosomal disorders, duplication/loss of genetic material, regional changes to level of a chromosome

- Single gene defects: mutations in individual genes that strongly affects phenotype/health - Multifactorial disease: influenced by genes AND environment, epigenetic modification, injury,

etc. as multiple possible contributors Information Maintenance and Flow CHAPTER 3 START

- DNA = “hard drive” - Replication: takes a mother helix and creates 2 daughter helices, [DNA-dependent, DNA

Polymerase] (not totally perfect b/c DNA pol. makes errors) - Transcription: production of mRNA, [DNA-dependent, RNA polymerase]

o mRNA gets modified more so it can be easily translated o mRNA 5’ to 3’ gives you N to C terminus in AA

- Translation: mRNA gets translated into AA sequence of polypeptide [ribosomes, tRNAs, etc] - Phenotype is at polypeptide level - Phenotype comes from transcription and translation, but not from replication b/c replication

doesn’t put forth any expression! - Ex) Pt w/ enzyme deficiency has mutation in DNA causes an aberrant codon to form in mRNA

misincorporation of AA in protein destroys active site of enzyme normally needed for life - Corruption @ DNA incorrect mRNA RNA Pol./ribos don’t catch error corrupted AAs - RNA pol doesn’t correct errors, just transcribes them b/c dumb - Ribosomes just translate them too - Reverse transcription: RNA info converted back into DNA [RNA dependent, DNA polymerase]

Yeast Proteome Central Dogma - Knocked out every single gene individually to see if “sociability” correlates to “essentiality” - Generally correlated—the more sociable you were, the more essential; but there were still some

that were popular but could be knocked out and cell would still live Central Dogma: Mom + Baby

- You have 1 phenotype composed of different traits or characteristics (not a hand phenotype, hair phenotype, ear phenotype separately)

- Organismal phenotype has components of anatomy, physiology, behavior - Organism organismal phenotype cell function gene network proteome genome - Alternative splicing: one gene can code for multiple proteins w/ distinct functions

Things We Expect to Find at a Genetic Locus - How to tell a region is genic?

1. Promoter: region of DNA w/ sequences in it that are binding sites for proteins known to regulate transcription (could also have Enhancers or Silencers before promoter)

a. Promotor binds proteins that help RNA Pol. mount close to the site of transcription 2. Start Site of Transcription: 3. 5’ UTR: region ribosome interacts with, nontranslated 4. Initiator Codon: AUG (mRNA), ATG (DNA), start site of translation! 5. Codons (exons, intervening sequences of introns that have to be cut out before translation)

a. Fully mature RNA will only have exons! 6. Terminator Codon 7. 3’ UTR 8. Polyadenylation signal

- Signals: Stop and go, promoters, enhancers, silencers, transcriptional stops - Genic information - 5’ Cap sites, splice donor and acceptor sites, codons, translation initiation and stop codons,

introns - Protein coding—would expect to see mRNA produced, translated to protein - Structural/catalytic RNA, rRNA, RNAse P, teloRNA etc can think of all these as genes even though

they don’t make proteins - Spliceosome can cut in middle of codons—doesn’t matter b/c reformed right after

Transcription (NUCLEUS) 1. RNA is transcribed (built) 5’ to 3’, based on a template DNA strand that is read 3’ to 5’. The “5’

UTR” refers to the transcribed UTR that is more 5’ on the RNA than the start site of transcription, but is actually located on the 3’ end of the DNA.

o The first nucleotide is +1. The first nucleotide UPSTREAM of it is -1. A Cis-regulatory site 20 base pairs upstream in the promoter region would be -20.

o Because RNA is synthesized 5’ to 3’, it matches up with a parallel DNA strand that is also 5’ to 3’ (sense strand), whereas its DNA transcription template is 3’ to 5’ (antisense strand)

2. A cap is added to the 5’ end of the RNA transcript 3. The 3’ end is capped w/ a polyA tail in the 3’UTR, which stabilizes the new RNA 4. RNA introns are spliced out so only exons remain 5. RNA is now mRNA and is transported out to the cytoplasm!

Translation (CYTOPLASM) 1. mRNA has 3-letter codons that code for different AAs (1st is AUG for methionine) 2. tRNA anticodon recognizes the mRNA codon and brings the correct AA into position along the

mRNA template 3. mRNA is translated into protein w/ help of tRNA and ribosomes 4. Stop codon signals the release of the polypeptide from the ribosome - Protein can be post-translationally modified w/ methyl, phosphates, carbohydrates

Genetic Locus Examples - B-globin gene:

o CAT box: binding site for transcription factor (in promoter region) o TATA: binding site for TATA binding protein (in promoter region)

- BRCA1 gene: o GC-rich region: associated w/ transcriptional activation (in promoter region

Lecture 4 Notes General Notes

- Kozak Sequence: region of mRNA that initiates the translation, contains the AUG sequence for methionine

- Raw mRNA transcript = 5’ CAP – UTR – Exons and Introns – PolyA site – 3’ end - Matured transcript = splice out introns, polyadenylate 3’ end - If you have a ribo in nucleus, it is being newly made in the nucleolar region - Translation does NOT occur in nuclear apartment; mRNA is shipped out for translation in cytosol - 5’ CAP sticks with the mRNA in the cytosol, proteins bind to the CAP to interact with the mRNA

during translation - AMPLIFICATION (way 1): if active promoter on DNA, allows 1 gene to produce TONS of mRNAs - AMPLIFICATION (way 2): mRNA sticks around in cytosol for multiple rounds of translation - Polyribosome: complex of mRNA and multiple ribosomes that chase each other and translate

tons of the same protein from one mRNA DNA Sense and Antisense

- Template strand (antisense, 3’ to 5’): strand that RNA uses as template and RNA Polymerase orients itself on based on promoters, etc.

- Nontemplate strand (sense 5’ to 3’): has the same sequence as the new sense RNA Promoter

- +1 = site of transcription: in future RNA, 5’ end would start here - TATA box (-20bp): TATAAAA most common just upstream of the start site of transcription - GC Box: GC-rich, targets for hypermethylation - CAT Box: for CAT Box binding protein to bind, followed by RNA Polymerase binds - Consensus: after surveying many genes, sequence is generally in this promoter region and is not

perfectly conserved, but is pretty similar - Stuff in the promoter region helps RNA Polymerase mount for mRNA transcription

TATA and Transcription Factors - Transcription factors bind sequentially and bring in more charged factors that attract the next

set of charged molecules + more TFs - Upstream stuff helps also RNA Polymerase figure out which DNA strand to use as template - TFIIF: helicase that pops open DNA and helps mRNA polymerase get access to the sequence of

nucleotides in DNA - RNA Polymerase holds helix open as it transcribes and moves twd 5’ end of DNA - Sequence of TFs helps mRNA Polymerase transition from a weak interaction with the promoter

region to a very strong interaction with open promoter complex and un-H-bound DNA helix (unzipped)

- TFs eventually dissociate and titin complex (mRNA Polymerase) chugs along DNA template strand and transcribes

More Transcription - RNA spends most time w/in solvent protection of RNA Polymerase until long - New RNA strand interacts w/ solvent environment (H2O) - H2O solvates RNA polyanion and pulls it into solution - As RNA Polymerase moves along DNA molecule, it doesn’t need help of TFIIF helicase anymore

and acts as its own helicase - Transcription terminator signal: portion of DNA that signals RNA Polymerase to stop transcribing

Supercoils - Supercoil: stretching open of DNA by RNA polymerase, which causes tension directly in front

and behind, translates through the enzyme

- We keep enough template strand open that we can transcribe it Interrupted Eukaryotic Genes

- Have to get nonsense info (introns) out of RNA (splicing) before we can use RNA for translation - 7-methyl guanosine (cap) is added to 5’ end of pre-mRNA

o 7-methyl guanosine has weird 5’ to 5’ triphosphate linkage to 5’ nucleotide base of mRNA, is added by a methyltransferase

o If uncapped, mRNA is hard to translate—ribosomes won’t recognize it, so you need to add a mix of methyltransferase and stuff for 7-methyl guanosine when making mRNA you intend to use

o Guanylyl Transferase adds the Cap, which Methyl Transferase later CH3s o Cap: binds to initiation translation proteins and small ribosome subunit

- Ribosome recognizes 5’ cap, associates with mRNA finds AUG - PolyA requires 3’ cleavage, polynucleotide adenylyltransferase extends 3’ end of mRNA with

PolyA tail, which helps maintain stability of mRNA - Spliceosome interacts with splice donor and acceptor sites, cause RNA of intron to covalently

bind to itself and remove itself from RNA sequence The Colinearity of Genetic Information

- Sequences of DNA sequences in RNA AAs in protein - Errors can travel straight down this line THEREFORE repair of DNA is v. important - DNA Polymerase has error correction

4 Stages of Translation 1. Activation: charging of tRNA with AAs (make sure tRNA gets the correct AA) 2. Initiation: binding of rb to mRNA, adds 1st AA (Met) at N-Terminus 3. Elongation: sequential addition of AAs to COOH end by many charged tRNAs 4. Termination: release factors bind when stop codons are found at ribosome’s A site; ribosome,

polypeptide, and mRNA disassociate Activation

- tRNAs are the physical bridge between RNA and protein worlds - tRNA: chimeric adapter molecule, nucleotide chain + AA; info bridge b/w mRNA codons and seq

of AA - tRNA structure:

o Free unpaired 3’ end = site of AA attachment (UNCHARGED if no AA attached, CHARGED if AA attached)

o Stem region: RNA-RNA helix / Open region o Anticodon (5’ to 3’): 3 nucleotides that form complementary H-bonding associations w/

complementary codon o Loops = 3D structure of tRNA, what aminoacyl tRNA synthetase uses in concert w/

anticodon to identify tRNA to associate specific AAs with their appropriate tRNAs o tRNA synthetases: associate specific AAs with their appropriate tRNAs—there are

specific synthetases for all AAs o L-shaped so synthetase can bind both the loops and stem at same time

Initiation - Need charged tRNA for methionine, interacts with IFs, small rb subunit - NO mRNA: bound structure associates with 5’ CAP and mounts initiator complex - 2 models: looping and standing

o Standing: initiator stands down until finds AUG within Kozak sequence, then pauses and IFs dissociate and large subunit of rb assembles

- Addition of Methionine at the peptidyl end is important, because it provides direction N-terminus anchor so other AAs can attach on at the C-terminus end; if not there, ribosome could not build polypeptide chain

Initiation: Importance of AA Structure - Side chains of AAs impart difference in charge, structure - AA structure determines orientation of the polypeptide chain - Polypeptides have N-terminal and C-terminal end - New AAs add onto the C-term end - Peptidyl Transferase of the large subunit produces the peptide bond - Ribosomes: made of rRNA (catalytic to peptidyl transferase) + proteins (charge-shielding and

making sure AAs associate) o Can think of ribosome as ribozyme, has a bunch of different parts that help do diff

things Ribosome Action

- E, P, A: sites composed of parts of large and small subunit, associated with 3 diff forms of tRNAs - E site: exit tRNA binding site (uncharged in picture b/c its AA is already part of the chain) - P site: “peptidyl” binding site, where AA chain is attached - A site: aminoacyl site, has charged tRNA w/ AA waiting to form a new peptide bond 1. Initiate complex forms, large subunit brought in 2. Initiator tRNA is at P site 3. tRNAs cycle in to sample codons on mRNA 4. Peptidyl transferase transfers AA at P site to the AA at A site thru formation of new peptide

bond and breaking of covalent linkage of tRNA/AA at P site 5. Elongation factors help the ribosome keep moving along mRNA toward the 3’ end 6. As ribosome moves, the A site becomes available with new codon, charged tRNA binds the A

site, and uncharged tRNA is released from E site (conveyer belt) a. Aminoacyl tRNA synthetases recharge tRNA by adding new AA to it

7. Ribosome encounters codon w/ no complementary tRNA (stop codon) 8. Causes A site to stay open too long, and proteins enter A site, release factors, cause release of

polypeptide chain 9. Dissociation of structure into RNA + lg subunit + small subunit + polypeptide chain + last tRNA

Genetic Code: Sequence of Codons in mRNA Dictates Sequence of AA in Polypeptide - Redundancy: multiple codons code for same AA

Ex. Mutations in Mito Genome - Presentation: Myoclonic epilepsy, small leg muscles, ataxia, CNS deafness instead of structural

defect, atrophy = problems with ENERGY INTENSIVE tissues, usually assessed w/ 2 diff tests - Gomori Trichrome Stain: differentiates muscle and nuclei from highly lipophilic/membranous

structures like mitochondria (proteins = green, nuclei = purple, mitos = red) - Cytochrome oxidase staining: to detect evidence of enzyme activity, tissue snap-frozen and

sectioned (brown = e-transfer can occur) - Image: mitos clumping and dying, cells full of mitos = no oxidative respiration - Conclusion: mitos heteroplasmic—some mitos have WT genomes, some have specific mutations

o Mutation = mitos have no lys tRNAs o Result when ribosome comes in and no lys tRNA, polypeptide just truncates, mitos fill

with truncated proteins, and mito cannot perform cell respiration

Lecture 5 Notes Overview: Goals, Approaches

- Before do any study, have to start with some complex DNA population - Specific amplication: DNA cloning (2 methods)

o Cell-based DNA cloning: creating a library (nonspecific) Need a suitable host cell (E. coli, yeast) Need replicon: plasmid, small bacterial cell Need way to bring foreign DNA and replicon together and transfer into host cell Scissors: DNAases and restriction enzymes Glue: DNA ligase

o Polymerase-mediated in vitro DNA cloning (PCR) (v. specific!) Need thermostable DNA polymerase Need sequence information enabling synthesis of specific oligonucleotide

primers - Specific detection

o Molecular hybridization w/ probes Need labeled DNA or RNA probe Need means of detecting fragments to which probe binds

Gene Cloning - Gene cloning: isolating and making copies of a gene - To make clones, use recombinant DNA technology - Uses: gene sequencing, mutagenesis, gene probes, expression of cloned genes - Restriction endonucleases/restriction enzymes: enzymes used to cut DNA (discovered naturally

in bacteria, bacteria’s immune defense against viral DNA!) - Restriction enzymes bind to specific DNA sequences and cleave DNA at 2 defined locations, one

on each strand - Endonucleases: bind to a site in the middle of DNA and cut it - Exonucleases: chew from a free 5’ or 3’ end

Basic Idea of Gene Cloning 1. Isolate your gene of interest (YGI) 2. Link YGI to bacterial or phage DNA 3. Introduce the recombinant DNA into a bacterial/phage host 4. Separate individual cells/clones 5. Make TONS of DNA for further analysis

Recombinant DNA Technology - We can isolate and manipulate fragments of DNA by cutting from one source (chromosomal

DNA) and pasting it into another molecule (vector DNA) - Recombinant molecule: DNA from genome + DNA from vector single molecule of DNA from 2

diff sources - Recombinant DNA molecules can be introduced into living cells that copy the recombinant DNA

and give us many copies of our gene of interest Example Restriction Enzyme Cut Site (EcoR1)

- Usually palindromic - EcoR1 cuts at the SAME point on both strands of DNA because of this palindrome, but leaves

overhang open for complementary base pairing (sticky ends) - Overhang ends would come together because of H-bonding between complementary bases if

the two halves were put in a bath, but the nick would still exist (need DNA ligase to heal) Using Restriction Enzymes

- Use EcoR1 to cut both human and replicon DNA at a certain site that gives fragments with identical overhangs

- Can then incubate cut human/bacterial DNA together until sticky ends find each other - Add DNA ligase to heal the backbone, and you get a human/bacterial recombinant

Problems with Using Restriction Enzymes - Restriction enzyme cuts EVERYWHERE it sees its cut site, so you can have a TON of different

weird fragments of human and bacterial DNA with same sticky ends 1. Human/human DNA can just re-anneal 2. Human/human weird fragments can re-anneal - Solution: try to use relative concentrations to force the product you want

o Usually use higher concentrations of vector and lower concentrations of target, so if target sticks to something, it’s more likely to be a vector when vector heals, you get a recombinant

o HOWEVER this means a ton of vector will just stick back together - SECRET SAUCE!: on vector DNA, use a phosphatase to take off 5’ Phosphate so vector can’t

anneal to itself and thus needs Phosphate from the target DNA - GOOD vectors only have one cut site per restriction enzyme

How to Clone a Piece of DNA 1. Cut vector and DNA with restriction enzymes 2. Ligate pieces together 3. Transform ligated DNA mix into bacterial host (E. coli)

a. Chemical transformation: use cations to beat up wall of bacteria swell bacteria w/ hypotonic solution so water rushes in and wall cracks recombinant DNA goes into cracks change osmolarity of sol’n and cracks reseal bacteria now has recombinant DNA inside it

b. Electroporation: use electrical current DNA around bacteria zap bacteria and electric current drives DNA into bacteria through cell wall

4. Plate transformed bacterial cells on agar and antibiotics to screen for clones/colonies w/ recombinant DNA

Chromosomal and Vector DNA - For CHROMOSOMAL DNA:

o Take your chromosomal DNA and treat with detergent and salt to remove membranes o Do a succession of phenol chloroform extractions to remove protein, membrane, other

stuff until you’re just left with nucleic acid - For VECTOR DNA:

o Usually way more vectors than bacterial genome present but they need to be purified o Protein anchors bacterial chromosome to membrane of bacterial cell, but plasmid floats

free (you want plasmid) o Use NaOH to kill cell and make “ghost” where genome is still anchored to membrane

but plasmids float free o Common cloning vectors take about 10K base pairs, bacterial artificial chromosomes

(BACs) can take 50-100K, yeast artificial chromosomes (YACs) can take up to 1 mill bps! o Vectors commonly derived from: plasmids, viruses

MUST-HAVES of Cloning Vectors 1. Ori (origin of replication): so that when bacteria replicates, plasmid replicates with it 2. At least one antibiotic resistance gene (as a selectable marker)

o If host is normally antibiotic sensitive, then any cell that grows in presence of antibiotic had to have picked up your plasmid (that confers resistance)

3. A number of unique restriction sites (where DNA fragments can be inserted)

Gene Cloning: Amplification

- Amplification of gene occurs 2 ways: - (1) vector gets replicated by host cell many times, generating a lot of copies per cell

o Plasmid origins are deregulated, compared to the bacterial chromosome - (2) bacterial cell divides every 30 mins, so overnight = many million!

DNA Libraries - DNA library: collection of thousands of cloned fragments of DNA - Genomic library: when the starting material is chromosomal DNA - Any individual colony in a library has only one piece of one chromosome in it

Genomic DNA Libraries - 4 diff copies of same chromosomal region of human chromosome - Restriction enzyme cut site is represented by red arrows - Partial digestion: use small amt restriction endonuclease to cut at SOME of the red arrows but

not all so that there are overlapping portions - Allows you to sequence small bits at a time and use the overlap to place where they should be

altogether Polymerase Chain Reaction (Kary Mullis)

- Generates billions of copies of a single molecule of DNA in a tube w/in hours without vectors or host cells

- Take DNA and use complimentary primers, use to make synthetic DNA replication reaction, primers will elongate and anneal if you add DNA ligase (boil/cool/elongate, repeat!)

What You Need to Do PCR - TARGET/Template DNA: contains region to be amplified - Sequence info for the target you want to study so you can make PRIMERS

o Primers: forward and reverse, hybridize to complementary strand o Complementary to sequences at the ends of DNA fragment to be amplified o Synthetic and about 15-20 nucleotides long

- Heat-stable DNA Polymerase: from Thermus aquaticus, could stand super high T - Nucleotides (ACGT), aka dNTPs - PCR Machine

Key to PCR: Thermal Cycling

- For each cycle: o 95° denature strands o 50° anneal primers o 72° polymerase extends/synthesizes strands o 95° separate strands, back to cycle

- Repeat sequential process of denaturing-annealing-synthesis for many cycles and you get TONS of DNA!

DNA Polymorphisms - Restriction site polymorphism: restriction sites that differ from human to human because of

slight differences or mutations in an individual’s DNA sequence

Lecture 6 Notes MUST-HAVES for Nucleic Acid Hybridization

- Probe: piece of DNA or RNA that is synonymous with the sense strand of DNA that the RNA matches up with, binds to its complementary antisense DNA strand in the right clone containing YGI, usually probe is tagged w/ fluorescent dye that glows when probe finds complementary strand

- Target: collection of heterogeneous DNA/RNA fragments BUT w/ desired fragment likely present, unlabeled

Types of Hybridization Probes - DNA Probe:

o Comes from cell-based DNA cloning or PCR o Starting material usually double-stranded o Labeled using DNA-polymerase to make the DNA strand + tag on the strand

- RNA Probe: o Very high affinity (DNA/RNA hybrid helix is STRONGEST) o Transcribed from insert DNA cloned in vectors o Starting material is single-stranded o Made and labeled using Run-off Transcription

- Oligonucleotide Probe o Chemically synthesized, usually only 15-20 bps long o End-labeled w/ polynucleotide kinase

How to Obtain a Probe! - If gene of interest has already been cloned, can use a piece of it as a probe - If not, use a probe that probably has a sequence similar to the gene of interest (rat B-globin is

probably similar to another rodent’s B-globin--yay for evolution!) How to Label a Probe!

- Strand Synthesis Labeling: o PCR: use to amplify and modify polynucleotide chain o Nick-translation o Random-primed labeling

- End-labeling: mostly for oligonucleotides - RNA labeling

Nick-Translation - Takes advantage of E. coli DNA synthesis - Used for oligonucleotides, <1000 bps

1. DNAse1 (E. coli enzyme) breaks single phosphodiester bonds, making nicks along backbone a. If low concentration and soak DNA in it, scatters nicks along helix but doesn’t fully

digest DNA b. If high concentration, breaks DNA into single nucleotides

2. Add DNA Pol. 1 to extend radioactively labeled nucleotides at the 3’OH end of the nick, while simultaneously chewing up DNA ahead of the nick (exonuclease + polymerase action)

3. Usually when DNA Pol 1 is extending nucleotides, dCTP* is tagged radioactively at alpha-P

Random Primed Labeling

- Used to make SUPER long primers, 1K – 10K bps 1. Denature double-stranded template with heat 2. Add mixture of hexanucleotides (unlabeled) that then bind to their complementary segments 3. Add Klenow subunit—modified E. coli DNA Pol w/o exonuclease—and dATP, dCTP*, dGTP, dTTP 4. Klenow subunit attaches nucleotide bases between hexanucleotides, including dCTP*

End Labeling using a Polynucleotide Kinase

- Use if you can’t use a fluorescent tag - Not as high signaling as dCTP*, but still useful for making many copies of labeled probe

oligonucleotide 1. Obtain an oligonucleotide with a free 5’-phosphate end 2. Use polynucleotide kinase to exchange one of the phosphates off ATP with an activated P32 3. Exchange the P32 with the “cold” free P on the 5’ end of the oligonucleotide 4. oligonucleotide probe with P32

Fill-in End Labeling - Not super important - Use to label 3’ ends of anything in your mix 1. Digest DNA with restriction enzymes to give 5’ overhangs 2. Incubate with Klenow subunit + dATP*, dTTP (or any mix of nucleotides as long as one is *) 3. Nucleotides fill in 3’ end 4. Use different restriction enzymes to cleave at internal sites and give fragments of DNA where 3’

ends are labeled

RNA Labeling Run-off Transcription - Use to make RNA-based probe (highest affinity against complementary DNA) - Can make LOTS of probe quickly - pSP64: vector

o amp: confers ampicillin resistance for when plasmid is taken up by bacteria for cloning o SP6 promoter: promoter for viral RNA polymerase o MCS: collection of individual restriction enzyme cutting sites (HindIII, EcoR1) o Ori: replication origin o Pvull: another restriction enzyme w/ cut site outside of where you want to insert foreign

DNA 1. Cut pSP64 at HindIII, EcoR1 sites; cut DNA insert at HindIII, EcoR1 sites 2. Put pSP64 and DNA together with DNA ligase to make a recombinant DNA/plasmid 3. Stick the plasmid in bacteria to create clones of recombinant plasmid

a. However, if add viral mRNA polymerase SP6 and ribonucleotides (UTP*, CTP, ATP, GTP) now, mRNA polymerase (SP6) would move through gene of interest and start translating all the rest of the plasmid we don’t want all this in our probe

4. Cut with another restriction enzyme (Pvull) to create linearized version of circular plasmid a. DNA helix is the same—it’s just a line instead of a circle now!

5. NOW, add SP6 mRNA polymerase + UTP*, CTP, ATP, GTP 6. mRNA binds at promoter, promoter fires, transcription happens through foreign DNA region

until mRNA polymerase runs out of template on the linear DNA and falls off a. you can have many mRNA polymerases running along the same piece of DNA at the

same time more amplification! b. 2 levels of amplification: 1 at cloning of plasmid, 1 at multiple mRNA pols on

Labels - Radioactive labels:

o Advantages: can detect small quantities (B particles given off easy to detect) o Disadvantages: health risks

- Non-radioactive labels: o Advantages: non-radioactive (less health risk) o Disadvantages: less signal

Ex) Fluorophores, chromogenic substances, chemiluminescent substances Autoradiography

- Affix DNA onto blot - Use probe on that blot—probe will attach to DNA - Put XRAY over and see where DNA is - Commonly used isotopes: 35S, 32P, 125I (usually for polynucleotide chain, not protein) - Also used: 3H, 14C

Blots - Usually use after you cut a DNA with restriction enzymes to get a bunch of fragments - Run those fragments through gel electrophoresis to sort them by size - DNA fragments (-) will stick to blot (+) so you just have DNA on a blot sheet - Can then use probe to find which fragments are complementary to probe

Nucleic Acid Hybridization Parameters - Depends on base pair complementarity between probe/target - Base composition, temperature, buffer conditions, ionic conditions - Good probe = known sequence (homogenous and complementary throughout its length), often

generated in vitro - Good target = heterogeneous mix of DNA/RNA fragment

Nucleic Acid Hybridization: 2 types of DNA/RNA helices - Homoduplexes: target hybridizes to self (target/target, probe/probe) booooo! - Heteroduplexes: probe complements + hybridizes to target w/ H-bonding (probe/target) want

Nucleic Acid Hybridization: Things That Determine STABILITY of Duplexes - Strand Length ↑length = ↑H-bonds det. Tm - Base composition G/C vs. A/T (G/C more stable b/c 3 H-bonds) det. Tm - Chemical environment buffer to stabilize pH, nonionic detergent to coat DNA + present nts - # of base mismatches human probe vs. rat target - Hybridization stringency: ionic strength + temperature

Melting Temperature - Tm is midpoint in transition from DS to SS DNA - ½ point between DS and SS is where complementary strands have a 50/50 chance of hybridizing

or melting - Melting just means breaking of H-bonds so DS DNA turns into SS

Base Mismatches - High hybridization stringency: when a single mismatch messes up an oligonucleotide probe

because oligonucleotides are so small to begin with - 20% mismatch - CANNOT use oligo probe, CAN use conventional DNA probe at reduced

hybridization stringency - High stringency = high T, low salt (DNA breaks apart easily) - Low stringency = low T, high salt (DNA doesn’t break apart easily) - SALT: cations in salt coat (-) backbone and prevent 2 backbones from repelling each other, great

for when you don’t have good complementarity

Dot Blots - USE: can survey complementary to any probe w/o having to do electrophoresis - Blot aqueous solution of denatured target DNA onto nitrocellulose filter, add probe, look for

reaction - Example: Allele-specific oligonucleotide (ASO) hybridization (ex. sickle cell anemia A->T) - Example: testing for gene mutations

Lecture 7 Notes Southern Blotting

- Use: to find (multiple) fragments of DNA in your genome that contain your region of interest 1. Digest human genome DNA with restriction enzymes to create a ton of diff. sized fragments 2. Separate fragments based on size using gel electrophoresis 3. DNA is still in helix form, so denature with alkaline (NaOH) to separate the 2 strands 4. Transfer from gel to filter paper blot 5. Add probe to find fragments with area of interest 6. Expose filter to x-ray film to see which bands/fragments attached to the probe! - ↑% agarose = pores in gel tight, small pieces give better resolution, large pieces get jammed - ↓% agarose = better size separation over wider range of sizes

Southern Blotting Uses 1. Can determine copy number of a gene in a genome (low stringency) 2. Can detect small gene deletions that can’t be detected by light microscopy 3. Can identify gene families 4. Can identify homologous genes among different species

Northern Blotting - Use: to identify a specific RNA within a mixture of many RNA molecules - Nucleotide on the gel is the EXPRESSED gene (or the RNA)

Northern Blotting Uses 1. Can determine if a specific gene is transcribed in a particular cell type 2. Can determine if a specific gene is transcribed at a particular stage of development 3. Can reveal if a pre-mRNA is alternatively spliced

Microarray Hybridization - Use: if you wanted to survey expression pattern of EVERY gene or look at transcription profiles

across organs, etc 1. Microarray contains a separate fragment of the entire human

genome (DNA) in each well 2. RNA is obtained from cells/tissue to be tested 3. Reverse transcriptase is used to transcribe the RNA into cDNA

for each tissue 4. The cDNA is tagged with a red (ex. cancerous cell cDNA) or green

(ex. standard cell cDNA) fluorescent dye and hybridized with the chip this cDNA can be a complete genome or just an oligonucleotide

5. If the cDNA hybridizes with the DNA in the well, the well fluoresces a certain color (red = cancer, green = standard cell, yellow = both), indicating that that gene fragment is active in the cancer, standard, or neither cell type

- Probe = every single known transcript in the human body, represented as small characters on a silicon chip

o SHH gene might be represented by 12 diff binding sites o 500,000 characters on chip array = 10-15 diff pieces of

all 25,000 human genes o Entire known genome is present on chip

Microarray Uses 1. Analyze differences in gene expression between 2 different samples

a. Ex. Cells at diff. stages of development

b. Cells w/ diff. characteristics 2. Analyze DNA variation across genome

a. Ex. Genetic testing (looking for mutations in known disease genes in an individual) Fluorescence in Situ Hybridization (FISH)

- Can use to visualize macromutations by using a mixture of chromosome paints (fluorescent DNA probes) such that each chromosome pair should only be a single color

o Allows you to see if parts of 1 chromosome are glomming onto another - Can see rearrangements, deletions, inversions use SKY and false color imaging - Combines Fluorescent Probe hybridization on DNA affixed to slides (usually via Lysine coating)

and Optical Microscopy with FLM - Uses: Pre-implantation screening of blastomeres, Cancer Cell Karyotyping, Downs Syndrome

Genotype Determination SKY Example: Colon cancer metaphase SKY Imaging

- SKY: Spectral Karyotyping - What’s wrong - Apc: part of kinetochore complex is mutant so spindle captures floaters and

chromosomes don’t line up on spindle apparatus - Chromosomes end up proliferating, forming weird Frankenstein chromosomes, things don’t

properly separate, mitosis is messed up Banding Patterns

- Unique staining patterns can be used to identify chromosomes and detect changes in chromosome structure

- G-banding: generated by Giemsa stain (Dark bands = more condensed chromatin) Numbering G bands

- Start at centromere out to telomere - Bands = local descriptions for where things are, but doesn’t tell number of base pairs

Character of Banding Patterns - Band numbers change with condensation (later = less bands) - Late Prophase: least condensed, most bands - Metaphase: most condensed, fewest bands - If you have a small feature you’re looking for, want to catch early before everything condenses

Studying Human Chromosomes - Fluorescence In Situ Hybridization (FISH): locates genes on chromosomes using a labeled probe - Chromosome Painting: causes whole chromosomes to fluoresce

o Use metaphase arrested cells o Put them through fluorescently activated chromosome sorter (FACS) o Isolate individual chromosomes that you use PCR to amplify to produce chromosome

specific probe libraries (allows you to paint whole chromosomes) - Comparative Genome Hybridization: label individuals’ whole genomes, turn their target into a

labeled target, and hybridize to array of BACs that represent individual human chromosomes o Typically you hybridize a male against a female array; female against a male array

Alu Sequence Banding - Derived from RNA portion of single recognition particle - Single recognition particle: ribonuclear protein that binds to potentially secreted proteins as

they emerge from rb and docks them to RER; underwent an eternal deletion and was reversed transcribed into DNA

o Has its own promoter region o By reverse transcribing this RNA into DNA, turned into mini-gene o Alu gene became retrotransposon and spread all over gene

- Uses repetitive DNA to generate high resolution banding patterns, by hybridizing Alu sequence specific probe to metaphase chromosomes

Comparative Genome Hybridization - Graph shows ratio (test DNA : control DNA) where control is typically a BAC that contains entire

human genome - Sample fluorescence : control fluorescence - Male patient is tested against a female control; female pt. is tested against a male control - Can see chromosomal dose (if there are 3 chromosomes at chromosome 18) and translocations

Chromosomal Abnormalities (2 “Classes”)

- Constitutional abnormalities: present in every cell of the body - Somatic (acquired) abnormalities: abnormalities only in certain tissues (like tumors), produces

“mosaics” - Genetic mosaics: individuals have different genetic makeup around entire body due to mitotic

defect o Trisomy 21 patients have some wild type cells, some trisomy 21 cells

Chromosomal Abnormalities (2 Structural Changes) - Changes in whole chromosome number (trisomy 21) - Changes in chromosome structure (translocations, deletions, duplications)

Variations in Chromosome Number - Euploidy (aka polyploidy): having the same number of each homologous chromosome (humans

normally have 2 of each chromosome; if you had 3 of each that would be euploidy) - Aneuploidy: variation in the number of a particular chromosome in a set - Mixoploidy: 2 or more genetically different cell lineages in an individual

How Ploidy Variations Occur - 2 haploid sperm can fertilize 1 oocyte (tetraploid) - Oocyte doesn’t have reduced nucleus (and is diploid) - Tetraploids – you usually see 2 copies of maternal chromosomes, 2 copies of paternal

Lecture 8 Notes Origins of Polyploidy

- Triploidy can be caused by: o 2 sperm + 1 oocyte o F meiosis fail = oocyte has a 2n nucleus o M meiosis fail = sperm has a 2n nucleus

- Tetraploidy can be caused by: o Normal M/F pronucleus fuses but later has mitotic catastrophe, causing tetraploid

Origins of Aneuploidy - Nondisjunction: chromosomes don’t separate properly during anaphase (in meiosis I, meiosis II,

or mitosis, but usually the first 2) - Meiotic nondisjunction can produce haploid cells that have too many or too few chromosomes

Nondisjunction in Meiosis I - What happens: failure to separate 2 homologs during anaphase I of meiosis I (reductional

division) - Meiosis II happens normally (equational division) - Result: 1 daughter cell has both copies of chromosome 2, 1 daughter cell has no copies of

chromosome 2 - Gametes: 2 (n+1), 2 (n-1)

Nondisjunction in Meiosis II - Meiosis I happens normally - What happens: in meiosis II, failure of sister chromatids to separate so 1 daughter cell gets too

many chromosomes, 1 daughter gets too little, and the other 2 daughter cells are fine - Result: n + 1, n – 1, n, n

Causes of Nondisjunction - Failure of spindle capture by kinetochore (like if only 1 kinetochore binds to a chromosome,

takes both chromatids into a single daughter cell) - Failure of machinery that orients kinetochore toward spindle apparatus

Examples of Aneuploidy - XX XX XX xx Diploid (2n) normal - XX XX XX Nullisomic (2n – 2) - XX XX XX x Monosomic (2n – 1) - XX XX X x Doubly Monosomic (2n – 1 – 1) - XX XX XX xxx Trisomic (2n + 1) NOT triploid, which would have 3 of each chromo. - XX XX XX xxxx Tetrasomic (2n + 2) - XX XX XXXX xxxx Doubly Tetrasomic (2n + 2 + 2)

Viable Autosome Aneuploids - Many more trisomics exist but are usually

spontaneously aborted before birth - Most XXX, XXXX, XXXXX females ARE fertile

but tend to have lower probability of bearing children because in meiosis, they would make aneuploid oocytes

- XYY trisomics appear totally normal male, prison study thought they were “supermales” and more aggressive but not necessarily true

- XXY, XXYY, XXXY Klinefelter males have problems with fertility, streaked gonads

Karyotyping Trisomy 21 - Trisomy 21 (Down Syndrome) will present with additional chromosome 21, but may not always

have free segregating homologs - Can also have pieces of 21 stuck to others (14, 15) - This is why it’s useful to use FISH to assess Downs and where the other pieces of 21 attached

Aneuploidy in Humans - Trisomy 13 (Patau Syndrome):

o most die early o double cleft makes nutrition hard b/c can’t suckle o solution is machine to seal entire face off o Characteristics: polydactyl, cleft lip/palate, small eyes

- Trisomy 18 (Edward Syndrome): o 80% of infants are F, because whatever region is tripled tends to be lethal to M (who are

spontaneously aborted) 20% of babies have a weird translocation so you want to do karyotyping of

parents AND child to see if the child inherited it from the parent or if the translocation was an accidental mutation just in the child

o 90% die w/in 6 months HIGH LETHALITY o Characteristics: rocker chair feet, clenched fist, low ears

- Trisomy 13, 18, 21 tend to be candidates for GENE SILENCING that aims to find out which tripled regions are most dangerous to cell and decrease their expression

Changes in Chromosome STRUCTURE - Deletion/deficiency: missing base pairs, usually distinct phenotypic characteristics - Duplication: gene DOSE is important! (ex. Charcot-Marie-Tooth) - Inversion: entire region of chromosome gets flipped - Translocation: piece of chromosome fused to another chromosome

Examples of Inversion/Deletions - Why breaks happen:

o H2O interaction o Radiation o DNA replication/recombination

- DNA gets repaired but not always perfectly - Paracentric Inversion: stable, can have no

phenotypic effect b/c swap can be outside of a genic region

- Interstitial deletion: individual is PARTIALLY ANEUPLOID, because only 1 gene with EF

- Pericentric Inversion: center inverted, can be fine as long as breakpoint doesn’t mess up a codon, ↓fertility

- Ring chromosome: if break fuses back on itself in ring!

- “para” around center - “peri” including center - Acentric: if centromere is deleted; acentric fragments are unstable because they don’t go

through mitosis or meiosis b/c there is nothing for the spindle apparatus to grab onto

Unbalanced Gamete Genotypes Caused by Crossover in Balanced Inversion Heterozygotes

Figure 5-10 Crossing over w/in inversion loops formed at meiosis I in carriers of a chromosome with segment B-C inverted (order A-C-B-D, instead of A-B-C-D). A, Paracentric inversion. Gametes formed after meiosis II usually contain either a normal (A-B-C-D) or a balanced (A-C-B-D) copy of the chromosome b/c the acentric and dicentric products of the crossover are inviable. B, Pericentric inversion. Gametes formed after meiosis II may be normal, balanced, or unbalanced. Unbalanced gametes contain a copy of the chromosome with a duplication or a deficiency of the material flanking the inverted segment (A-B-C-A or D-B-C-D).

- Inversions are usually seen in heterogeneous individuals bc inversion chromosomes are rare in

the population—you could get one from your mom, but would most likely have WT from dad - Inversion heterozygote: 1 normal homolog + 1 inverted homolog - Dicentric: chromosome has 2 centromeres = tug of war - Acentric: bad b/c you have no way to interact w/ spindle apparatus - Paracentric gametes are normal + balanced + 2 inviable (dicentric + acentric)

o Inviable because dicentric causes tug of war, and acentric has no way of interacting w/ spindle

o Dicentric/acentric are duplicated and simultaneously deleted - Pericentric gametes are normal + balanced + 2 unbalanced

Child w/ Pericentric Inversion

- gamete is aneuploidy because it’s missing p25-pter on one chromosome - Chromosome 3 duplication there is a duplication on chromosome 3 - q21-qter, deletion p25-pter q21-qter is duplicated, while p25-pter is deleted (like AB∙CA) - inv(3)(p25q21) inversion on chromosome 3 with breakpoints at p25 and q21

Figure 5-9 Array CGH analysis of chromosome abnormalities. A, Detection of a partial duplication of chromosome 12p in a patient with an apparently normal routine karyotype and symptoms of Pallister-Killian syndrome. (Sex chromosome data are not shown.) B, Detection of terminal deletion of chromosome 1p by array CGH in a patient with mental retardation. C, Detection of an approximately 5 Mb de novo deletion of chromosome 7q22 by array CGH in a patient with a complex abnormal phenotype; this deletion was originally undetected by routine karyotyping.

Potential Problem: Can’t See Differences - What if you can’t detect differences using Giemsa staining, Alu banding, Comparative Genome

Hybridization? All you know is that brother/sister both have it. o You’ve discovered a single gene mutation within family

- Single gene mutations are hard to discover using this technology, has to be thousands of chromosome differences