Biol Exam #2 study Guide
Biol Exam #2 study Guide Bio 219
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This 8 page Study Guide was uploaded by Sophia Valla on Thursday October 6, 2016. The Study Guide belongs to Bio 219 at West Virginia University taught by Lima Huebert in Summer 2016. Since its upload, it has received 8 views. For similar materials see The Living Cell in Biology at West Virginia University.
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Date Created: 10/06/16
BIOL 219 Study Guide Exam #2 Human Genome ● Haploid ○ 3.2 billion bases / 20,000 genes on 23 chromosomes Reannealing Curve estimates size and complexity of the genome ● Isolate DNA (cut into short pieces) ● Make to same conc. ● Denature by heat (renature/ hybridize) ○ Small genes reanneal faster Cot = initial DNA conc. And time Less repetitive = more complex More repetitive= less complex (reanneals faster) # of genes are NOT proportional to complexity OR genome size Fluorescent In Situ hybridization (FISH) ● Looks at # of copied DNA regions ● Majority of the genome is random repeats ○ Mostly transposons ■ Jumping genes ■ LINES / SINES ● Copy and paste (RNA transcriptase RNA reverse transcriptase DNA) ■ Cut and paste (nuclease and ligase) ○ Satellites = 10^7 base pairs ○ Minisatellites = 10^5 base pairs ○ Microsatellites = 15 base pairs (up to 100 bp total) (forensic use) Genomes Changes ● Duplication ● Deletion ● Insertion ● Inversion DNA Hereditary 1. Replication 2. Must have structure or function info 3. Change overtime for evolution Griffiths 1928 ● Bacteria can transfer info through transformation ● Combined nontoxic strain mixed with killed toxic strain = live toxic strain Avery, McCarthy, MacLeod 1943 ● DNA (not proteins) can transform a cell ● Break see what's broken for transforming agent (reverse genetics) Hershey, Chase 1950 ● Showed that DNA is the genetic material not protien ● Inserted bacteriophages (DNA and proteins) only DNA entered bacteria %A + %C = %T + %G (not all equal) Watson and Crick 1953 ● Rearranged nucleotide pieces till they properly fit makes DNA helix DNA/RNA composed of nucleotides ● Sugar ○ OH on 3’ C ■ OH on 2’ C (RNA) ■ H on 2 ‘C (DNA) ● Phosphate ● Base ○ A/G = purines ○ C/T = pyrimidines Stranded DNA B DNA ● major/minor grooves ● 2nm width ● 3.4 nm length = 10 bp ● Binding proteins major grooves of B DNA (better access at major grooves) DNA is 1.8 m long Nucleosome existence supported by microscopy DNA Protection Assay Isolate DNA Add small amount DNase Separate DNA out Run on gel (separates by size) Nucleosomes (1st level chromatin structure) ● 4 proteins x 2 8 total ○ (H2A, H2B, H3, H4) ● 150 bp of DNA = wrapped 2 x 75bp ● Tails are polar (lysine and arginine) ○ Binds DNA with positive proteins DNA Organized in Interphase (higher order chromatin) ● Nucleolus ○ Moderately repetitive ○ Code rDNA for ribosomes ● Heterochromatin ○ Tightly packed ○ Near nuclear membrane ● Euchromatin ○ More open (accessible) ○ Closer to center of nucleus Nucleus Eukaryon Genomic DNA Separates genomic activity from rest of the cell (transcription & translation) Nuclear Envelope Connects directly to ER Lamins (mechanical strength) Binds directly to chromatin Depolarize in mitosis Nuclear Pore Complexes (NPCs) Many different nucleoporins Inside ring is hydrophobic Lets mRNA leave nucleus Transport Proteins Importin going in (Nuclear Localization Signals (NLS)) Ran GTP OR cargo Exportin going out (Nuclear Export Signals (NES)) Ran GTP AND cargo G Protein (diff. Allosteric conformations) ● GDP inactive ○ Guanosine exchange factor ○ Conc. high in cytosol ● GTP active ○ GTPase activating protein (turns off) ○ Conc. high in nucleus Meselson and Stahl (proves semiconservative DNA) ● Conservative ○ 1st gen : N15 only /N14 only ○ 2nd gen: N15 only/ N14 only ● Semiconservative ○ 1st gen: N15/N14 hybrid ○ 2nd gen: N14 only / N15 N14 hybrid ● Dispersive ○ 1st gen: N15/N14 hybrids ○ 2nd gen: N15/N14 hybrids Bidirectionality E Coli in H3 replicate twice Replication fork Replicates toward fork Multiple origins of replication (bubbles) increased replication Semi Discontinuous Okazaki segments labeled Ligase glues segments together To test: break ligase SSB keeps from reannealing DNA helicase breaks H bonds with ATP Primase makes RNA primer Topoisomerase relieves tension (breaks phosphodiester bonds in backbone the glue it back together) DNA Polymerase 3 binds RNA/DNA complex extends growing strand with DNA (does most of the work) DNA polymerase 1 takes out RNA primer Fills with DNA in lagging strand (discovered first) Leading Strand ● DNA pol 3 continues until it hits the other replication for at the other side of the circle or end of a linear chromosome ● DNA pol 1 has to only replace 1 RNA primer with DNA Lagging Strand ● DNA pol 3 dissociates from DNA when it reaches RNA primers ● RNA primers targeted by DNA polymerase Looping keeps it connected pol enzyme moving in same direction Keeps both pol connected so they copy simultaneously Eukaryotic Replication ● More pol with different functions ● RNA primer removal by RNase ( not polymerase) DNA Proofreading ● DNA pol 1 ( 3 enzymes in 1) ○ Polymerase activity ○ 5’ to 3’ exonuclease (cleaving RNA primers) ○ 3’ to 5’ exonuclease (fixes mistakes) Detects mistakes by base pair angles End Replication problems in Linear DNA Telomeres repeating sequence on ends of chromosomes Telomerase enzyme made of RNA and protein ● Protein and RNA template ● Extends template strand ● Always adds same sequence ● DNA pol 1 extends new strand from primer 12 different base substitution can occur in DNA Transition (within groups) Transversion (purine switches with pyrimidine) Changes in bond angles (mismatched bp) ● Pol 1 stops ● Mismatch fed through nuclease site ● Cleaved during synthesis (S phase) ● Fixed in G1 and G2 ● Recognized in transcription Thymine dimer formation by covalent bonds inhibits pol and stops replication Depurinations cleaves out a purine (guanine) Base Excision Repair (point mutation) Depurination Cuts out nucleotides not base pairs polymerase fills it in ligase glues it For pyrimidine dimers Surveillance method : cuts out 1 twist (10 bp) Polymerase fills in ligase glues Double Strand Breaks ● Nonhomologous end joining (translocations) ○ Ku recognizes ends that are broken ○ DNAPKcs pulls ends together ○ DNA ligase IV glues ends together Phenotypic Variation (only germ line can be passed on) ● Point mutations ● Double strand breaks ● Translocation ● Insertion / deletion ● Transposons Replication Amplification (bacteria) Will replicate and piece of circular DNA (plasmid) with correct properties PCR ● 94℃ (DNA opens) ● 56℃68℃ (DNA reannels) ● 72℃ (polymerase activity) Reagent specifies the region that will be amplified (primers) Mendelian Inheritance Linked alleles from different genes on same chromosome are not separated during meiosis Recombination can unlink alleles 1. Nuclease causes a double strand break 2. Strand invasion new template to fix break 3. DNA repair by DNA polymerase 4. Ligase glues together 5. Knots forms that moves along strand a. Rotates around to make Holliday junction b. Cleaved vertically c. Ligase glues (recombination product) d. Cleaved horizontally e. Ligase glues (mostly recombination) FISH identifies location of genes on a chromosome Determine Sequences Chain termination based on DNA replication mechanism Similar to PCR Reagents DNA pol Primer Buffer dNTPs= 90% ddNTPs (no 3’ OH) =10% (terminates chain) Next generation sequencing really parallel Ligate known sequence on end Use for PCR Builds clusters (same sequence) Sequencing by synthesis (color coded) Lon Torrent ● Pass each nucleotide in order ● Record when H+ is released ● After condensation rxn Sequencing problems ● DNA pol can’t sequence long pieces Cells → isolate DNA → fragment → size selection → placed in segment plasmids → build primers 1. Sequence one piece → FISH to find overlap → sequence next piece 2. Shotgun sequence a. Sequence all of it at the same time → use computer to find overlap → generate one long chain Template sequence works well for repetitive regions Primer walking works well at ends of chromosomes Guess and check works well if you have many sequences Studies with Gnome Tracing ● 29 Mammals ○ Comparative genomic study ○ Catalogs diversity and identifies keys to evolution ● Microbiome studies ○ Sequence organisms to catalog diversity and identify microbes ● Hapmap ○ Catalog human diversity ○ Haplotypes set of alleles that are linked together ○ Correlated to geography 1000 Genome and GWA (genome wide association study) ● Choose individual to represent medical diversity ● Sequence genome ● Find patterns with relation to traits ● Simple version 23 and Me Manhattan Plot Probability of association vs. position on chromosome Synthetic Genomics Smallest genome Recombination and replication Transformation Large scale Jurassic Park Small scale gene editing Prokaryotes ● Circular chromosome ● Haploid ● Single celled / no nucleus ● Loops attached to plasmid membrane ● No histones packaged as nucleoid New Genetic Info in Bacteria 1. Conjugation a. Physical contact between bacteria b. Transfer is undirectional c. Plasmids are transferred rolling circle replication d. HFR cells high frequency of recombination i. F (fertile) plasmid integrates into bacterial genome ii. Transfer some genome iii. High recombination due to homology iv. Location of f plasmid integration is random 2. Transformation a. Environmental DNA b. Transporter channel protein and helicase i. Spins DNA into cell ii. component able to make up free DNA ( transporter , CaCl2) c. Gene transfer i. Adding partial diploid / dominant allele expressed ii. Replacing 2 crossovers, based in homology d. Plasmids small extrachromosomal pieces of DNA i. Transferred by transformation 3. Transduction a. Viral intermediate b. DNA from previous host → injects DNA → 2 crossovers → allele swap c. Lytic or lysogenic i. Fast little time for host genome incorporation d. Lysogenic i. Inject DNA ii. Transposons integrate in genome iii. Hibernates until stressed iv. Excised phage will make new virus v. Good gene transfer Complementation ● Bacteria that lack in some genes can be mixed with bacteria that has the gene offspring can grow on minimal media Compare Donor and Recipient to map genome ● Test which bacteria grow on minimal media ● How often two pieces are transferred together depends on distance ○ High frequency = 1 allele transferred ○ Medium frequency = 2 allele transferred ○ Low frequency = 3 allele transferred Transformation Put on petri dish and give straight DNA Conjugation Have them touch in a U shaped tube lets phages pass
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