BIOL 3451 1st Edition Lecture 13 Outline of Last Lecture I. 9.2 Knowledge of Mitochondrial and Chloroplast DNA Helps Explain Organelle HeredityII. 9.3 Mutations in Mitochondrial DNA Cause Human DisordersIII. 9.4 In Maternal Effect, the Maternal Genotype Has Strong Influence during Early DevelopmentIV. 10.1 Genetic Material Must Exhibit Four CharacteristicsV. 10.2 Until 1944, Observations Favored Protein as the Genetic MaterialVI. 10.3 Evidence Favoring DNA as the Genetic Material Was First Obtained during the Studyof Bacteria and BacteriophagesVII. 10.4 Indirect and Direct Evidence Supports the Concept that DNA Is the Genetic Materialin EukaryotesVIII. 10.5 RNA Serves as the Genetic Material in Some VirusesIX. 10.6 Knowledge of Nucleic Acid Chemistry Is Essential to the Understanding of DNA Structure X. 10.7 The Structure of DNA Holds the Key to Understanding Its FunctionXI. 10.8 Alternative Forms of DNA ExistXII. 10.9 The Structure of RNA is Chemically Similar to DNA, but Single StrandedXIII. 10.10 Many Analytical Techniques Have Been Useful during the Investigation of DNA and RNAOutline of Current Lecture I. 10.10 Many Analytical Techniques Have Been Useful during the Investigation of DNA andRNAII. 11.1 DNA is Reproduced by Semiconservative ReplicationIII. 11.2 DNA Synthesis in Bacteria Involves Five Polymerases, as well as Other EnzymesIV. 11.3 Many Complex Issues Must Be Resolved during DNA ReplicationCurrent LectureI. 10.10 Many Analytical Techniques Have Been Useful during the Investigation of DNA andRNA- The Svedberg values are not absolute (32S is not 2x 16S); shape plays a part in velocity. - Heat/other stresses can cause DNA to denature Hyperchromic shift: used to determine melting temperature Figure 10.17These notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.- Molecular hybridization (Figure 10.18) Denature, RNA strand hybridized to create hybrid Denaturation and renturation of nucleic acids essential for this Helped advance molecular evolution and organization of DNA in chromosomes, and increased understanding of gene transcription Do transcriptome studies (determining which genes get turned on/off/up/down); good for cancer- Fluorescent in situ hybridization (FISH) Uses fluorescent probes to monitor hybridization Used to identify the chromosomal location of a DNA of interest1. Ex: DNA specific centromeres of human DNA (Figure 10.19)2. Also be done by asking human genome- Reassociation kinetics: analyzes rate of reassociation of complementary single DNA strand Provides information about size and complexity of genomic DN from an organism- Nucleic acid electrophoresis: separates DNA and RNA fragments by size; smaller fragments migrate through gel faster than large fragments (figure 10.20)II. 11.1 DNA is Reproduced by Semiconservative Replication- DNA strands can serve as templates to be complimented- Called conserved when old duplex is conserved- Three models of DNA replication are possible: Conservative1. Original helix conserved, and 2 newly made strands come together Semiconservative1. Each replicated DNA molecule has one “old” strand and one new strand Dispersive1. Parental strands are dispersed into two new double helicesa. Similar to semiconservative (1 old, 1 new), but there is some variance- Meselson and Stahl (1958): used (N15 and N14) E. coli to demonstrate DNA replication is semiconservative in prokaryotes Each new DNA molecule consists of one old strand and one newly made strand1. Semiconservative and dispersive: everyone would be 14.52. The heavier (denser) the molecule, it changes strand outcome- Taylor-Woods-Hughes: used broad bean and showed DNA semiconservative in eukaryotes (not as important as Meselson and Stahl)- DNA replication begins at origin of replication Where this occurs, strands of helix unwound, creating replication fork Bidirectional; so there are two replication forks- Length (section of DNA) that is replicated from origin is called a replicon- Entire bacterial chromosome constitutes 1 replicon (6.6 million) (LOOK AT)- We (humans) can replicate DNA faster than E. coli (if it wanted) by: Creating 23 chromosomes, not just 1 Having a hundred origins on a single chromosomeIII. 11.2 DNA Synthesis in Bacteria Involves Five Polymerases, as well as Other Enzymes- DNA polymerase: catalyzes DNA synthesis and requires DNA template and all four deoxyribonucleoside triphosphates  DNA polymerase is dependent on template, also require primer, and has to go from 5’ to 3’ (add 1 nucleotide at a time) Figure 11.7, 11.8- DNA polymerase I, II, III can elongate a primer using a template, but not initiate DNAsynthesis (Table 11.2) All three have 5’ to 3’ synthetic activity (work towards 3’) 3’ to 5’ exonuclease activity 1. Used for proofreading newly created DNA and removed and replace incorrect nucleotides2. DNA polymerase I shows 5’ to 3’ exonuclease activity; can chop and replace gaps behind (Nic translation (motion))3. Table 11.2 (the last one: 5’ to 3’ talking about the Nic translation-whichonly I can do) DNA polymerase III does 5’ to 3’ polymerization essential in vivo  Have to have something very accurate, otherwise you would have too many mutations DNA polymerase III is complex enzyme (holoenzyme) made of 10 subunits whose functions are known (more, but we don’t know function)1. Table 11.3 Holoenzyme and some other proteins at the replication fork from a complex replisomeIV. 11.3 Many Complex Issues Must Be Resolved during DNA Replication- Seven key issues must be resolved DNA replication: Unwind helix (requires energy to break hydrogen bonds) Reduce increasing coiling generated during winding (requires gyrase enzyme) Synthesis of a primer for initiation Discontinuous synthesis of second strand (Okazaki) Removal of RNA primers  Joining of gap-filling DNA t the adjacent strand Proofreading (also need additional scanning to fix after DNA polymerase leaves)- DnaA binds to origin of replication and responsible for initial steps in unwinding the helix  1st one we need,  Figure 11.9 (curved part is origin)1. B, C begins unwinding right next the origin- Subsequent binding of DnaB and DnaC further opens and destabilizes the helix These proteins (helicases) need energy normally