MCDB 310 1st Edition Lecture 10Outline of Last Lecture I. Beta Chitin (revisted)II. Glycoconjugatesa. Proteoglycansb. Glycoproteinsc. LipoproteinsIII. Nucleotides and nucleic acidsa. Fundamentals of nucleic acidsb. Structure of Nucleic acidsc. DNAd. RNAOutline of Current Lecture I. Structure of RNAII. Nucleic Acid Chemistrya. Denaturation/renaturation of DNAb. Using duplex hybrids in the labc. Mutations in DNAd. Other nucleotide functionsIII. DNA based technologyCurrent LectureThese 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.I. Structure of RNA (continued from last lecture)a. Hairpins, large loops (fragile), smaller loops (more stable)b. Having some RNA molecules broken/dysfunctional is not as harmful in cells as mutated DNAc. Transfer RNA: one end binds to mRNA and one end that binds to amino acidsi. All 20 different kinds of tRNA only differ in these ends (specific to anticodon/amino acid combo) the rest of the tRNA is just designed to keep these two ends very far apart (can be very similar or very different between 20 different tRNAs)ii. Ribozymes: Ribosomal enzymes (all enzyme material applies to this)II. Nucleic Acid Chemistrya. DNA at room temperature in aqueous solutions is viscousb. DNA denaturation (melting) happens at 80 degrees Celsius or pH extremes i. Hydrogen bonds between bases are broken and base stacking is disruptedii. DNA is unwound and strands separate (either partial or complete)iii. This is only a change in CONFORMATIONiv. This kind of denaturation does not cause the DNA to lose its functionc. Renaturation (annealing)i. If some small parts of two DNA strands are still attached (anneal in one rapid step)ii. If strands are completely separated:1. 1st step: Slow (strands randomly collide, trying to make base pairs)2. 2nd step: Fast (strands zipper together)d. Each species has a characteristic denaturation temperaturei. The higher the GC content, the higher the denaturation temperature (GC has 3 H bonds, AT has two H bonds)ii. Can use this difference in denaturation temperature to purify/identify different DNA moleculese. Denaturation/Renaturation reveals evolutionary historyi. Melt pieces of human DNA and they anneal over a few hours (use restriction enzymes to cut DNA at specific places in sequence)ii. But when you melt human DNA AND mouse DNA1. Some sequences anneal over a few hours2. Relative number of Hybrid Duplexes (when one strand from one species binds to one strand from another species) means more Sequence Homology3. This method is reversiblef. Using Hybridization in the Labi. Southern Blot: Identifying DNA (same as Western blot for proteins)1. Separate DNA segments by size in a gel (adarose-larger pores)2. Transfer separated segments to membrane and electrophorese it out3. All of the DNA segments are then on the surface of the membrane4. Add in radioactively-tagged segments of another species DNA5. Let it sit6. Test for the amount of radioactivity: the radioactively tagged sequences that bind to the sequences of the original segments create Duplex Hybrids (more radioactivity, more duplex hybrids)7. Result: non-purified DNA (can separate 50-75 bands)ii. Polymerase Chain Reaction: an extension of Southern Blot1. Heat to denature the DNA2. Bind oligonucleotide primers (specific to sequence of interest) andadditional bases3. Add heat stable DNA polymerase4. Duplicate the DNA sequence between the primers5. Repeat the above steps 20-30 timesiii. DNA Microarrays: identifies gene sequences in a whole genome or plasmid1. Exact same technology as Southern Blot, but with this technique we know exactly which sequences are present in each resulting banda. Cut DNA using restriction enzymesb. Put each different DNA segment in a tubec. Perform PCR on each tubed. Robotic machine puts multiple samples of each tube on the array in a specific spote. Can add different species DNA with a fluorescent tag and wash the rest away, resulting only in the duplex hybrids that can be identified by the fluorescent tagsi. Can do many different tags (ie-one for actin and one for myosin)ii. Looking at colors tells us which of these form duplex hybrids2. Quantitative, and it may identify several sequences on the same array at the same time3. This technique can resolve thousands of sequences of DNA (much better than southern blot)g. Permanent Changes in DNA (mutations)i. Deamination: loss of amine groups (sometimes by UV light)ii. Deamination of Cytosine  Uracil1. This causes a problem in DNA2. There are enzymes that can repair this mutation (happens a lot every day, 1E7 times per cell per 24 hours)iii. Deamination of A to hypoxanthine and G to Xanthine1. Can be caused by nitrous acid and bisulfite (prevent bacterial growth in food industry)2. Happens often in humans (1E5 times per cell per 24 hours)iv. Depurination: removal of the whole purine residue1. Guanine residue in DNA is removed2. There is nothing that will form a base pair3. Causes a bulge in the DNA and loss of the codonv. Causes of these mutations:1. UV light: thymidine (pyrimidine) dimers on same DNA stranda. Forms a covalent bond between two Ts in same strand of DNAb. Happens with either a C5/C6 linkkink (two bases comingcloser) or C4/C6 linkc. Can be fixed by enzymes2. Ionizing radiation and X-Rays3. Oxidative damageh. Long Term DNA Damage (preservation)i. What is the half-life of DNA?1. If we find a mammoth with some preserved marrow, can we get enough DNA out of it to sequence it?2. Estimated by looking at Moa (extinct bird) bones that after 521 years half of the DNA will have degraded3. This estimate indicates that all bonds in DNA would be broken in 6.8 million years4. However, groundwater, temperature extremes, or microbes present can reduce this estimate5. Old Seed Germination:a. Found some seeds under permafrost in Siberiab. Took the DNA and were able to grow a plantc. The seeds were 32, 000 years old (much more stable than estimated by the other lab)i. Other Nucleotide Functionsi. Chemical Energy Sources (example: ATP)1. Phosphates covalently linked the 5’ hydroxyl of a ribonucleotideii. Cofactors:1. Adenosine is present in all of the important cofactors2. This is because adenosine does not participate in the chemical reaction of phosphate group transfer3. The removal of this adenosine reduces the function of a cofactoriii. Adenosine can fit into a Nucleotide-Binding Fold (place on