The power of bacterial genetics is the potential for studying rare events
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1 The types of experiments that a geneticist can do are dictated to a great extent by the model organism that s/he is working with The power of bacterial genetics is the potential for studying rare events 1
2 Let s start with some human genetics Achondroplasia is a completely penetrant, autosomal dominant disorder characterized by disproportionate short stature -- the arms and legs are short compared with the head and trunk. More than 80% of the people who have achondroplasia are born to parents of normal stature. Findings from molecular studies have shown that these sporadic cases of acondroplasia are almost always caused by mutations inherited from the father. The occurrence of achondroplasia is higher for older fathers: 50% of children with achondroplasia are born to father older than 35 years old. There is no association with maternal age. TAKE A GUESS AT THE FREQUENCY PER LIVE BIRTHS OF ACHONDROPLASIA 2
3 An estimate is derived of the mutation of achondroplasia based upon the accumulated data of recent newborn studies in four cities. In a total of 242,257 births, seven infants had mutant achondroplasia, the diagnosis being confirmed radiologically in all but one. From this, the rate of mutation of the normal to the achondroplasia allele is calculated to be 1.4 X 10-5 mutations in the achondroplasia gene per gamete = 1 mutation per 69,216 copies of the gene In Spain, Martinez-Frias et al. (1991) found a frequency of achondroplasia of 2.53 per 100,000 live births. 2.53/200,000 gene copies = 1.5 X
4 Decoding mutation jargon The mutations just discussed are single base substitutions. The most striking is achondroplasia, in which 153 of 154 analysed cases are due to a glycine to arginine substitution at codon 1,138. The mutations are in the transmembrane domain of the fibroblast growth factor receptor 3 (FGFR3). Of the 153 mutations, 150 were guanine to adenine transitions and three were guanine to cytosine transversions of the same nucleotide. This means that all the cases of achondroplasia are due to changes in one nucleotide a nucleotide with the highest known mutation rate (about 10-5 per generation). There are mutations at other sites in this gene, but the phenotypes are different. The achondroplasia mutations are all at a CpG nucleotide pair, known to be a mutation hot-spot. What do you know about CpG islands? 4
5 ** BLAME ASSESSMENT ** It has been established that fathers are the source of all achondroplasia mutations the probability of having an affected offspring increases explonentially with the father s age Figure 3 Relative frequency of de novo achondroplasia and Apert syndrome for different paternal ages. The ordinate is the ratio of the observed number of mutations (O) to the number expected (E), if all paternal ages are associated with the same frequency of mutation. The blue line gives the actual data; the red line is the best-fitting exponential curve. (Figure adapted from ref. 4.) 5
6 What fundamental difference in the way human male and female gametes are produced could explain the fact that fathers are the primary source of many of new gene mutations? 6
7 Cell divisions during oogenesis and spermatogenesis. S, stem cells; G, gonial cells; M, meiotic cells. The total number of cell divisions in the life history of an egg is 24. In males this depends on the number of stem-cell divisions, which is greater in older males. 7
8 The germline mutation rate in human males, especially older males, is generally much higher than in females, mainly because in males there are many more germ-cell divisions Why is number of cell divisions correlated with mutation rate? 8
9 The power of bacterial genetics is the potential for studying rare events The Mutagenesis lab is a great illustration of this: STRAINS USED IN THIS EXPERIMENT: E. coli B CGSC 5365 = wildtype strain Spontaneous forward mutations affecting antibiotic sensitivity and ability to utilize lactose E. coli B CGSC 5365 mutagenized with EMS (at WWU) Induced forward mutations affecting antibiotic sensitivity and ability to utilize lactose as the sole energy/carbon source lac - derivatives of CGSC 5365 Spontaneous reverse mutations restoring ability to utilize lactose as the sole energy/carbon source 9
10 BEFORE FRIDAY: Carefully review mutagenesis handout and work up table and/or flow chart summarizing dilution series (plural) and platings. CT will check notebooks for this NOTE that you are responsible for working up the protocol for measuring spontaneous reverse mutations in the lac gene: PART IV: MEASURING LAC - LAC + REVERTANTS FRIDAY MARCH 2 Before lab on March 2, work out a protocol for measuring the frequency of spontaneous reversion mutations in a culture of lac - cells. In your notebook, specify the type of media that you will be using and how you will carry out the plating portion of the assay. Each pair of students will examine one of the lac - strains. 10
11 What is the Biggest inadvertent Biology Experiment Ever Performed: that with the grandest scale? 11
12 12
13 This table illustrates evolution in action: what three requirement for evolution by natural selection does this table illustrate? Science 293: 1786 Sept. 7, 2001 See article on antibody resistance: Science 288: 792 May
14 What are the three requirements for evolution by natural selection? 14
15 Science 293: 1786 Sept. 7, 2001 These resistance genes didn t rise out of nowhere Where do the antibiotic resistance genes come from? 15
16 Mechanisms of AR: Pumps to get rid of compound Target of A is mutated so it is not sensitive A is Degraded by product of AR gene ACQUIRED VS ADAPTIVE SEE ARTICLE 16
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