double stranded DNA: AGAGAGAGAGAGAGAGA TCTCTCTCTCTCTCTCT The 2 hydrogen bonds of A pair exactly with the 2 H bonds of T The 3 hydrogen bonds of G pair exactly with the 3 H bonds of C As the two strands split apart, two new strands are synthesized from deoxyATP, deoxyGTP, deoxyCTP and deoxyTTP "Template directed synthesis" analogy to a photographic positive bound to a photographic negative Semi-conservative replication: Meselson-Stahl Experiment Matthew Meselson has made many other discoveries, and was also chiefly responsible for persuading the US (via Kissinger) to abolish its germ warfare program in the early 1970s. (which the US really DID abolish. I believe) Russia signed a treaty to quit, but then Gorbachov used US foreign aid money to build a huge anthrax factory in the Urals, from which a leak then killed at least 64 Russians. Meselson and his wife played key roles in uncovering this. Each single strand of DNA and RNA (unless they are circles) has a three-prime end and a five-prime end Ribose sugar is linked to phosphates at 3'carbon and 5'carbon. But new subunits can only be added at the 3' end! (for some reason) DNA synthesis enzymes are called DNA polymerase; The first such polymerases to be isolated (mid 1950s) later turned out to be for repair of damage, not regular copying. Then other polymerases were found, all of which added bases only at the 3-prime ends of the new chain. It was expected that the "real" DNA copying enzyme would be able to add to both strands simultaneously! But none exists! A MAJOR PARADOX!! This paradox was eventually solved by some strange discoveries The true method is continuous elongation from 5' to 3' to form the "leading strand" & "Okazaki fragments" are polymerized on the "lagging strand" each also in the 5' to 3' direction and are then linked together by "ligase" enzyme. There are also RNA primers, for starting each fragment! Topoisomerases are enzymes that allow coiled DNA strands to spin (by cutting one of the strands) and even for DNA strands to pass through each other (Type II topoisomerases.) A few antibiotics work by blocking procaryote topoisomerases. ("ciprofloxacin"; as was used to treat anthrax, and other diseases) (but in a germ attack, at least 80% of those exposed will probably die, even with the best treatment, including prior vaccination) PCR = the Polymerase Chain Reaction is a useful method for making millions of copies of DNA and specifically just of DNA containing certain base sequences. Heating DNA, to nearly to boiling temperature (95 C.) causes the two strands to separate. Then cool, and add specific "primer" single-strand sequences. Then add deoxyATP etc. and DNA polymerase enzyme which will add copy in 3' direction from bound primers then heat again, to separate new strands. repeat cycle, repeat again 1, 2, 4, 8, 16, 32, 64, 128, 256, 500+, 1000+, 2000+, 4000+, 8000+, 16,000+ 32,000+, 64,000+, 128,000+, 256,000+, 512,000+, one million + (20 cycles) 30 cycles-> one billion 40 cycles-> one trillion When this method was first invented, you had to add more enzyme for each cycle, because the boiling destroyed it. But then certain bacteria from Yellowstone boiling springs were used as a source of heat resistant DNA polymerase. PCR has been used to amplify mitochondrial DNA from bones of Neanderthal cave men and amplify DNA from blood, hair, etc. at crime scenes, etc. Sometimes in DNA copying, the wrong base gets added. Unless it is removed and replaced, that will be a mutation. Ultraviolet light and many chemicals can damage DNA, sometimes causing adjacent bases to bond to each other, etc. forming "thymine dimers", among other abnormalities. Special sets of enzymes find such damage, find "wrong" bases and cut them out and put back the correct bases. Without these special enzymes, mutation rates are much higher Some people have mutations in the genes for repair enzymes: "Xeroderma pigmentosa" causing their rates of skin cancer to be thousands of times higher. Incidentally, one of the main functions of the G1-S checkpoint is to detect DNA damage, and not allow DNA copying to begin until all this damage is repaired. Or, when the DNA is too badly damaged, to cause self destruction of the cell ("apoptosis") (self-digestion) Most anti-cancer drugs damage DNA, often by cross-linking. Mustard gas also damages DNA severely, by covalent bonding. "Nitrogen mustards" are important group of anti-cancer drugs, designed to damage DNA in the same way as mustard gas. Nerve gas acts differently: by covalent bonding to -OH in serine at the active sites of many enzymes; esp. acetylcholinesterase Mustard gas was developed in WWI; but not used in WWII. Iraq killed ~50,000+ Iranian soldiers with mustard gas and ~10,000+ Kurdish civilians with nerve gas. RNA copies are made by copying one strand of DNA; with the RNA having the same base sequence as one DNA strand (but with U instead of T; thymine is just methyl-uracil) & has the complementary base sequence to the other strand. "Transcription" means making such RNA copies of DNA Do not confuse the words transcription and translation; These words have special meanings in molecular biology, and both are used A LOT! Please get used to them having the following meanings: transcription = RNA synthesis translation = Protein synthesis RNA synthesis is catalysed by enzymes called RNA polymerase (addition of more subunits at the 3' end) Only certain special parts of DNA get copied (and only one of the two strands in any one region) This is controlled by preferential binding of RNA polymerase to just certain base sequences pribnow box, etc. "promoter regions" Only the DNA "downstream" of promoters gets transcribed. (downstream being toward the 5' end/ direction, of the DNA) To illustrate this fact: "Pseudogenes" are lengths of DNA where the base sequence codes for some actual protein, but has no promoter sequence upstream of the pseudogene. If you somehow inserted a promoter sequence upstream of a pseudogene, then an RNA for it would get transcribed. And what ought to happen if you inserted promoter sequences in random locations in an organism's genome? Three main categories of RNA (each coded for by its own genes = DNA sequences) Transfer RNA: each binds to its own amino acid; so there are glycine t-RNAs, alanine t-RNAs etc. Each containing a 3-base sequence: the anti-codon (For a glycine t-RNA, the anticodon is CCC) Ribosomal RNA (ribosomes are million+ molecular weight enzyme complexes where proteins are made from amino acids.) Messenger RNA (that codes for the amino acid sequence of proteins, according to the genetic code) in the sense that if a messenger RNA has the base sequence AAA, that means add a glycine in the protein at that point. To everybody's great surprise, (most) eucaryote genes have introns, which are intercalated regions of many 10s or 100s of DNA base pairs that will NOT get translated, because these sequences will be cut out of the RNA transcripts! Nobody understands why they exist! Procaryotes don't have introns. The genes for a few specific proteins (histones are an example) don't have introns. And some species have more and bigger introns in their genes that other species. Nobody expected or had predicted that introns would exist, and there is not yet any one favorite hypothesis to explain why they evolved. But as soon as researchers started sequencing genes, introns were immediately obvious. Introns get copied into the m-RNA precursor, but then spliced out before the m-RNA goes to the cytoplasm. The parts of the RNA that remain in the messenger are called "exons". (nothing to do with the oil company!) For many proteins, several different versions get made, because of different processing of their m-RNA Other aspects of "processing" of messenger-RNA: "Caps" and "poly-A-tails: Questions that you should now be able to answer: 1) What are the raw materials from which DNA is synthesized? 2) If these raw materials were radioactively labeled, then after a double stranded DNA is copied to make two copies of this DNA, then where would the radioactive label be found? a) In one strand, but not the other? b) Equally in both strands? c) In neither strand? d) Sometimes one and sometimes the other? e) None of the above? 3) What was confirmed by the famous "Meselson Stahl experiment? *4) Where would the radioactivity be after several more rounds of DNA strand duplication? *5) In the preceding questions, we assumed that the radioactive label was in either the ribose, or in the purines and pyrimidines, of the deoxyATP etc. But what if the radioactive phosphate had been used? Explain. 6) What are the names of the two different kinds of ends of DNA and RNA strands? *7) In double stranded DNA, the 3' end of one strand is next to what part of the other strand? 8) How can double stranded DNA uncoil and re-coil when being copied? *9) Are there any ways to inhibit this uncoiling in germs, but not animal cells? For what purpose might you want to do that? 10) When double stranded DNA is copied, are both strands copied the same way? Or what? 11) Does the wrong base ever get added in DNA synthesis? 12) Does the addition of the wrong base necessarily result in a mutation? What else might happen? 13) How can mutations in certain genes result in changing the rate of mutation of all genes, including themselves? 14) Where did the heat-resistant DNA polymerase come from? 15) For what specific method is this heat-resistant enzyme especially useful? 16) How is it possible to make large amounts of DNA, with the same base sequences, from tiny amounts of blood or other cells found at a crime scene? Could DNA be duplicated a billion-fold? *17) A few years ago, several students in Biology 52 turned out to believe that Darwinian evolution worked by having "bad" mutations edited out of the DNA, while "good" mutations were not edited out! What process had these students misunderstood? *18) To put the question another way: when enzymes selectively "fix" abnormal base sequences, can they do this based on the phenotype (= effect) of the mutation, or something else? 19*) Can this mechanism distinguish between mutations with good effects and mutations with bad effects? 20) How is repair of DNA damage related to one of the cell cycle checkpoints? 21) What are pseudogenes? What makes them different from genes? 22) Why wouldn't a mutation in a pseudogene have any effect on the phenotype of an organism? 23) Why would DNA repair mechanisms be just as likely to repair damage to DNA in pseudogenes as in actual genes? 24) How could mutations in the DNA near a pseudogene cause this gene to be transcribed into a messenger RNA? 25) What part of the gene for a transfer RNA would need to be mutated in order to cause it to bind to messenger RNA at the wrong codons? 26) How are introns related to the ability of some eucaryote genes to code for proteins of several different sizes, with slightly different properties? *27) Guess why ribosomes are (barely) visible as dots by transmission electron microscopy. 28) Besides splicing out the introns, what two other changes are made in "processing" eucaryote messenger RNA 29) In embryonic development, the control of gene expression is usually by "transcriptional control", but sometimes by "translational control". Figure out what these terms must mean. If an egg contains stored messenger RNA, that doesn't get used to make protein until after fertilization, which type of control is that? What about when messenger RNA for a given protein only gets made in red blood cells, or some other cell type? 30) What's wrong with the statements, sometimes read in the newspaper, that DNA is converted into RNA and RNA is then converted into protein? What really happens? What is the misunderstanding?

 

back to syllabus