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University of Toronto Scarborough
Biological Sciences
Monica Sauer

Lec 13: DNA – The Stuff of Heredity - to investigate a crime scene, collect blood sample and look at DNA - What does it mean to say something is genetic? o It’s hereditary o Comes from parents o Can’t hide it - How many nucleotides are in DNA? A: p. 262 - Where is DNA found? A: nucleus How do we know DNA is hereditary material? (p. 188) - Hershey & Chase Experiment  Phage (bacteriophage): viruses that infect bacteria, have protein coats (viruses leave their coat when they enter a cell) o Allowed to grow on a) bacteria containing sulphur and b) phosphorus o Sulphur is component of proteins and would label the protein coat o Phosphorus is component of DNA and would label DNA o THEN, allowed viruses to affect completely normal bacteria o Hypothesis: If hereditary material is protein, then the hereditary material the phage transmit to the bacteria will contain radioactive sulphur; alternatively, if the hereditary material is DNA, then the hereditary material the phage transmit to the bacteria will contain radioactive phosphorus *p. 261 o Methods: some bacteria containing the phage genetic material were blended to “shake off their coats” / sep. coats from material inside the cells  Hershey & Chase analyzed the cell surface coat-containing part for radioactivity o Results:  Progeny phage that had radioactive sulphur did NOT contain radioactivity  Phage that had radioactive phosphorus containing DNA did contain radioactivity! o Conclusion: DNA is hereditary material b/c the hereditary material that was transmitted was labeled with P which labels DNA DNA Structure - Contains sugar phosphate backbone - Phosphate group attached to 5’ end - Sugar attached to 3’ end - Nucleotides composed of: phosphate group, sugar, base: purines (Adenine & Guamine) and pyrimidines (Thymine & Cytosine) - Base Pairs: AT / GC - Watson & Crick discovered structure w/ help from Wilkins and Franklin *bold = Noble Peace Prize o Franklin did xray defraction of DNA molecule and found that when looked upon from above, appeared egg-shaped/helical o DNA = spiral staircase Does DNA replicate semi-conservatively? - Semi conservative: each strand produces its own template and that served as template for next model - Conservative: each strand was its own template but just in new DNA; new strands paired together and old strands paired together to create double helix - Dispersive: Little fragments all the way thru replicated so mixture of new and old Question: Does DNA replicate semi-conservatively? Methods: used E. coli + heavy N15 and allowed one round of DNA replication o Grew them on N14 (light Nitrogen) o Spun bacteria with CsCl o Heavier stuff at bottom, light at top Results: DNA from N15 settled to bottom o Middle, there was mixture of both o After 2 rounds of replication, 2 bands: 1 at top (light) and 2 in the middle Conclusion: Semi-conservative DNA Replication: 1. Helicases unwind the DNA right where it uncoils 2. Primases make a short RNA molecule that starts a new strand of DNA 3. Polymerases add nucleotides to leading strand (continuous fashion) and lagging strand (discontinuous and produces small pieces called Okazaki fragments) extending the DNA 4. Single-stranded binding proteins (SSBs) stabilize the unwound DNA; keep them uncoiled 5. Topiosomerases are enzymes that prevent twisting of the unwinding DNA 6. Ligases (TO TIE) tie the Okazaki fragments together as discontinuous replication continues, sealing the nick between the 2 lagging strands. Leading & Lagging Strand - Anti-parallel fashion - Origins of replication (ori) occur in many places along the DNA in eukaryotes, allows for tremendous speed of replicating DNA Taq polymerase (Thermus aquaticus DNA polymerase) - Enzyme that permits the bacteria to have DNA replication at extremely high temperatures - Hot springs - DNA replication: 5’  3’ direction PCR Reaction Must-haves: - polymerase, primer, sample of DNA, nucleotides, proper temp (EXTREMELY high heat) - Use of DNA: e.g. DNA finger printing, forensic analysis Lec 14: Chromosomes and the Stuff of Heredity - Chromosome: coloured bodies, are the genetic units divided by mitosis Quiz: - How many chromosomes do humans have? - What is the composition of chromosomes in a skin cell and a sperm cell? - How does mitosis change the # of chromosomes? - Telomerases: enzymes involved in adding DNA to ends of some eukaryotic chromosomes o May signal inhibition of cell division o In differentiated cells, they are inactive; In cancer cells, they are active Where & When does DNA replication occur? - A: nucleus, interphase What are chromosomes made of? - DNA, which is wound around histone proteins and those together are called nucleosome o Histone proteins help DNA be packed into the chromosome o Important b/c when prepping a karyotype, first must denature proteins and then use Giemsa reagent to stain DNA, which produces characteristic G-light or G-dark patterns for each chromosome  G-dark regions have higher DNA density  Density of active genes is higher in G-light bands o After the Giemsa reagent is used, can align chromosomes according to size and position of centromere - Karyotype Prep: o Blood sample taken and added to growth medium that contains chemical which stimulates mitosis o Cells allowed to grow for 2-3 days @ body temp o Colchicine added to arrest cell division at metaphase o Arrested cells transferred to centrifuge tube o Centrifugation concentrates the cells at the bottom of the tube o Addition of hypotonic salt solution causes them to swell up and move apart o Lysed cells are prepared, fixed and placed on microscope slide o Microscope slide is photographed, enlarged to see chromosomes, cut out and paired/matched/aligned horizontally according to centromeres Fruitfly (Drosophila Melanogaster) Chromosomes o # of chromo = 4 pairs o How is sex determined? XX females, XY males Chromosome Numbers (p. 164) - Aneuploidy: the chromo # is NOT a multiple of the haploid chromosome number o E.g. female, only 1 X chromosome = Turner’s syndrome - Haploid: one copy of each type of chromosome in the nucleus e.g. bacteria cells - Diploid: 2 copies of each type of chromosome in the nucleus e.g. human cells, rhinos - Polyploidy: multiple copies of each type of chromo e.g. strawberry, banana o Why would you want a polyploidy crop plant?  Makes really BIG fruits - Haplodiploidy: bees; males are haploid, arise from unfertilized eggs  Females are diploid How is sex determined in mammals? - Turkey (meleagris gallapavo) chromosomes: o 39 pairs of autosomes o 1 pair of sex chromo o Bird sex chromo = WZ and ZZ - Butterflies/moths: o Female is heterogametic sex - Grasshoppers: o Diff # of chromo that determine sex o Female = even # (2) and male = odd # (1) o Presence of X in female but absence in males  first hint that maybe genes are found on chromosomes - Zebra fish: o NO sex chromosomes o Watch them mate - Oak gall: o Wasp lays eggs on one of these leaves and something on the egg itself triggers plant hormones that stimulates cell division; cell divides and grows into a gall form and larva hatches out of egg and chows down on plant material Mitosis (p. 170) OVERVIEW - Chromosomes are genetic material divided by mitosis - Interphase: DNA replicated, chromoso
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