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Lecture 9 Gene structure and function: a historical overview 1. Proof that DNA is genetic material 2. Initial insight into the nature of mutations 3. Bacteriophage crosses 4. Intragenic recombination 5. Cis-trans test for complementation 6. Double helix Streptococcus pneumoniae Experiments of Griffiths in 1928 showed that bacteria can be genetically altered by some chemical substances (a discovery of bacterial transformation). R type (rough colonies) – nonvirulent bacteria; S type (smooth colonies) – virulent bacteria In 1944, Avery, MacLeod and McCarty showed that virulence is associated with DNA Electron micrograph and diagram of bacteriophage T2 Life cycle of virulent phages T2 or T4 T4 phage genome: ~300 genes, 1700 bp E. coli genome: 4,254 genes, 4.6 x 106 bp In 1952 by Hershey and Chase proved that genetic material is DNA Hershey would subsequently share the 1969 Nobel Prize in Physiology or Medicine for his work in discovering the properties of DNA. But Marta Chase (1927-2003), who served as Hershey's lab assistant during his experiments and whose name appears on the paper, was snubbed. She earned her Ph.D. in 1964 from the USC. A series of personal setbacks through the 1960s ended her career in science. She spent decades suffering from a form of dementia that robbed her of shortterm memory. Alfred D. Hershey 1908-1997 Phosphorus is found in DNA, while sulfur in proteins The experiment that shows that DNA, not protein, is genetic material The injected genetic material contained phosphorus, therefore, it is DNA, not protein the structures not for memorization Selection of E. coli resistant to phage T1 (tonr mutants) Spread bacteria (108) over the plate and incubate overnight at 37 oC Mix bacteria (108) with phages (109) and incubate overnight at 37 oC bacterial lawn (uniform growth over the entire surface) only a few individual tonr colonies will grow What is mutation? a stable and inheritable physiological adaptation? a cell tons a cell tonr a spontaneous change? allele tons allele tonr Luria-Delbrück experiment (1943) (also called the Fluctuation Test) demonstrates that new mutations causing resistance to phage T1 arise spontaneously (in the absence of any selection) rather than being a physiological change in response to selection. adaptation spontaneous change In 1952 Joshua and Ester Lederberg demonstrated that auxotrophic mutations arise spontaneously and are then selected (replica plating) Some new terms: Mutation rate e.g.: 3 x 10-8 mutations per cell division Mutation frequency e.g.: 5.7 x 10-7 mutations per cell these are actual data for tonS tonR new mutations Radiation (X-ray and other types) cause mutations Hermann J. Muller The Nobel Prize in Physiology or Medicine 1946 ionizing radiation often cause chromosomal breaks at high doses 1 rad = the dose causing 100 ergs of energy to be absorbed by one gram of matter; 1 krad = 1000 rads (1 erg = 10−7 joule) FYI not for memorization chemical mutagens predominantly cause point mutations FYI Phage colonies (plaques) grown on the lawn of E. coli cells phage plaques E. coli lawn Phage phenotypes: • appearance of plaques • resistance to drugs • host specificity • various biochemical features commercially available Petri dish full of E. coli cells A cross of bacteriophages Parental phages: small turbid (h- r+) large clear (h+ r-) Recombinant plaques RF m.u. = % Total plaques Recombinant phages: small clear (h+ r+) large turbid (h- r-) recombinant phages Seymour Benzer in early 1950-ies at Purdue Univ. Bead theory of genes: By the early 1950-ies that was the prevailing view on the structure and arrangement of genes on chromosomes. Benzer proved it was wrong some time later Later in CalTech Phage rII mutants could grow only with E. coli strain B mutant phage: no plaques wild type phage: E. coli strain K (nonpermissive) E. coli strain B (permissive) Phage T4 mutant rII E. coli B large E.coli K no plaques w/t (r+) small small Intragenic recombination in rII mutants rII gene rII gene rII1 rII1,2 rII2 w/t Can grow only on E. coli B Can grow on both E. coli B and K A rare inside-the-gene) crossover between two different rII mutants generates a double mutant and a w/t phage Cross two mutants by coinfection a permissive culture of E. coli B cells, obtain phage progeny Plate the progeny on a lawn of non-permissive K cells Plate the progeny on a lawn of permissive B cells Count the plaques. Only w/t progeny can grow = half of all the recombinants (double mutants cannot grow on K cells) Count the plaques. All kinds of phages can grow = total progeny RF = Rec. / Total Determine RF = 2 x w/t % total RFmin = 0.01% But how many genes are there in the rII gene region: just one or more? A complementation test rII gene region with many individual mutations that were accurately mapped 1. If there is just one gene, then no two mutations will complement each other when two mutant phages coinfect the same cell: no rII gene product (an enzyme or other protein) is produced by either mutant rII1 Enzyme rII2 No phage growth (no cell lysis) = no complementation 1. If there are two genes, then mutations in different genes will complement each other when two mutant phages coinfect the same cell: each mutant is deficient with respect to one product, but when combined they will provide all the products necessary rII1 rIIA Enzyme A rIIB Enzyme B rII2 rIIA rIIB Phage growth (cell lysis) = complementation Note: no recombination is involved here! The progeny phages are the original mutants! A test for complementation in phages - despite the host being non-permissive! - the original mutants Complementation tests for determining the units of function in the rII region of phage T4; the nonpermissive host E. coli K12 () is infected with two different rII mutants. (a) Complementation occurs. (b) Complementation does not occur. A test for complementation – aka „cis-trans test‟ Cistron = gene cis-configuration: mutations on the same chromosome trans-configuration: mutations on different chromosomes How can DNA perform genetic functions? It should satisfy the following criteria: - be able to store genetic information; - be able to change (mutate) - be able to pass this information from generation to generation. The answer to these questions required knowledge of the detailed chemical structure of DNA. Erwin Chargaff analyzed the nucleotide composition of DNA from many organisms and found the regularities in molar concentrations of nucleotides: A=T G=C A+G = C+T = 50% but A+T ≠ G+C Rosalind Franklin and Maurice Wilkins studied X-ray diffraction patterns of DNA The famous “photo 51”, shown to James Watson by Maurice Wilkins without Franklin's knowledge, was the critical evidence that led to the confirmation of the postulated double helical structure of DNA, (see more on: http://en.wikipedia.org/wiki/Maurice_Wilkins) Their data suggested that DNA is a long molecule and represents some sort of a spiral or helix with periodicity of a 3.4 nm. Franklin even suggested it is a double helix. In 1953 Watson and Crick put these two sets of data (Chargaff‟s and Franklin-Wilkins‟) together and produced the model of DNA threedimensional structure: the famous double helix How can DNA perform genetic functions? It should satisfy the following criteria: - be able to store genetic information: The information is stored in the form of nucleotide sequence. A genetic code is used to define amino acids by threenucleotide combinations (triplet codons) in DNA. - be able to change (mutate): The information may change (a mutation may occur) by a change of nucleotide sequence. - be able to pass this information from generation to generation The information is passed on to progeny by the mechanism of semiconservative replication of DNA, wherein each strand of DNA is used as a template for synthesis of another strand, as was first realized by Watson and Crick, who wrote... Semiconservative replication of DNA The most famous understatement in the histrory of science: “...It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.” Watson and Crick (1953) Nature, 171:737-738 "for their discoveries concerning the molecular structure of nucleic acids and its significance for information transfer in living material" Rosalind Elsie Franklin 1920-1958 A 1962 photo shows Nobel Prize winners (from left): Maurice Wilkins (medicine), Max F. Perutz (chemistry), Francis Crick (medicine), John Steinbeck (literature), James Watson (medicine) and John C. Kendrew (chemistry). Not pictured are winners Linus Pauling (peace) and Lev Landau (physics).