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Terminology for chromosomal rearrangements: (used in connection with Muller's mutant allele category tests to manipulate gene dose) Df = deficiency = deletion.

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Presentation on theme: "Terminology for chromosomal rearrangements: (used in connection with Muller's mutant allele category tests to manipulate gene dose) Df = deficiency = deletion."— Presentation transcript:

1 Terminology for chromosomal rearrangements: (used in connection with Muller's mutant allele category tests to manipulate gene dose) Df = deficiency = deletion Bulge in the synapsed polytene chromosomes of a heterozygote shows what is deleted Fig A physical map of the genome in Muller's day (used to reduce allele dose)

2 Terminology for chromosomal rearrangements: (used in connection with Muller's mutant allele category tests to manipulate gene dose) Dp = duplication (insertional vs. tandem) (used to increase allele dose) Df = deficiency = deletion (used to reduce allele dose) Bulge in the synapsed polytene chromosomes of a heterozygote shows what is deleted (or duplicated) what is deleted Such rearrangements helped workers locate genes on the polytene chromosome map.

3 Inversions also have helped locate genes on the Drosophila polytene chromosome map Inversion breakpoints (In)

4 fertilized egg (embryo) 1st instar larva 2nd instar larva 3rd instar larva Pupa ( metamorphosis ) 1 day 2 days 4 days adult ™ ¢ (reproductive in ~2 days) XX XY If a gene functions at different times to do different things, temperature shifts of ts mutants can reveal those various functions Drosophila life

5 determination of the “temperature-sensitive period” (TSP) by single temperature shifts up or down point (time) in development when temp. shifted embryo larva pupa adult ultimate phenotype all OK all mut restrictive unshifted permissive unshifted shift rstrctv to prmsv shift prmsv to restrv TSP start : when begins drop TSP end : when wildtype TSP single

6 determination of the “temperature-sensitive period” (TSP) point (time) in development when exposed to rstrctv. temp for some fixed period of time (relatively short for high resolution). embryo larva pupa adult ultimate phenotype all OK all mut restrictive unshifted permissive unshifted TSP Temperature pulses (double-shifts) are more definative: shift from prmsv to restrv AND BACK P P R gene function needed: 12

7 Temperature-sensitive mutant alleles help us recognize (“dissect”) a gene’s pleiotropy (multiple, “unrelated” functions) point (time) in development when exposed to the restrictive temp. embryo larva pupa adult ultimate phenotype all OK all mut restrictive unshifted permissive unshifted two TSPs: time of function 1 2 Consider if: needed for an adult behavior essential for development & viability vs.behavior viability low behavior abnormal mutant animal survives, allowing us to see the behavioral abnormality

8 (1) What kinds can we make? (categories) (2) How do we make them? (mutagenesis) (3) How do we find them? (mutant screens & selections) Mutations (changes in DNA): the lifeblood of genetic analysis (4) Why bother?

9 Spontaneous mutations: Factors affecting Mutation Rates Why do they occur at the rate they do? DNA mistakes happen (nothing is perfect) (1) It costs energy and time to avoid making mistakes …proofreading of DNA replication (2) It doesn’t pay to be perfect DNA synthesis and repair genes determine the spontaneous mutation rate Consider: Natural selection optimizes spontaneous mutation rate redwood trees vs. E.coli MEASURE: changes per gene per: “mutator” mutations: increase spontaneous rate gamete cell division generation

10 Mutagens: agents that increase mutation rate Chemicals: (1) base analogs incorporated in place of normal, then misbehave 5-BrU (5-bromouracil) (Fig. 7-10, learn for MCATs) incorp like T, generally pairs w/ A), but tautomerizes to pair with G (2) base modifying agents modifications change pairing rules EMS (ethylmethane sulfonate) Ethylated G pairs with T Ethylated T pairs with G GC TA>CG >GC>GC >AT >G*>G*>GT>GT >*T>*T (3) intercalating agents slip in between bases noncovalently cause additions and deletions acridine orange induces replication error induced transition or transversion?

11 Mutagens: agents that increase mutation rate Chemicals: (1) base analogs (2) base modifying agents (3) intercalating agents (4) original root beer (sasparilla root) (5) urine from smokers organic all-natural carcinogen histidine - histidine + reversion rate Ames test for mutagens [p224. Fig 7.16] Salmonella typhimurium requiresdoes not require auxotrophprototroph used a panel of different molecularly defined mutants

12 (1) …generally easier to induce germline mutations in males than in females. in sperm: little to “distract” the mutagen from DNA With chemical mutagens:

13 (wt) after 1st zygotic division subsequent rounds of replication do not invariably cause errors: With chemical mutagens: (2) …often progeny from mutated sperm are genetically mosaic for new mutations only one of the two complementary bases in sperm are modified: GC -> G * C -> G * T & GC in sperm G * T -> G * C & AT wt mutant Question: if an animal is mosaic for the new mutant base pair, under what (developmental) circumstances will that new mutation not be transmitted to the progeny? Answer: if the mutant base pair doen't end up in germline stem cells.

14 Radiation (the first experimental mutagen discovered) non-ionizing (lower energy) ionizing (higher energy) UV light photochemical reaction glues adjacent thymines together in cis: A G G C C T C T T C A T C C G G A G A A G T T A G G C C T C T=T C A T C C G G A G A A G T DNA repair machinery called out: light-dependent repair (accurate) excision repair (error prone) replication block X-rays,  -rays, cosmic rays (pity the poor shoe salesmen of yore)

15 Radiation (the first experimental mutagen discovered) ionizing (higher energy) X-rays,  -rays, cosmic rays generates free radicals:chemical bull in the china shop …can induce double-strand DNA breaks important to repair at any cost!! …but what repair template available? -- information on the sister chromatid, but only after DNA replication -- information on the homologous chromosome in diploid organisms If none can be found (or found in time), just stick the DNA together blindly. Hence,ionizing radiation causes just about every molecular category of genetic change imaginable. (double helix shown, not sister chromatids)

16 Reading: “Transposable genetic elements move from place to place in the genome” pp (Chp 14) Also: p548 (insertion sequences in bacteria) A practice problem set dealing with mutant categories is available on the website. Answers will be posted next Monday. Lecture schedule: Wed 3/14: genetic screens Fri 3/16: sensitized screens Mon 3/19: genetic mosaics in screens (hence: no RNAi and one less sex lecture)

17 Radiation (the first experimental mutagen discovered) non-ionizing (lower energy) ionizing (higher energy) UV light excision repair (error prone) photochemical reaction glues adjacent thymines together in cis: A G G C C T C T T C A T C C G G A G A A G T T A G G C C T C T=T C A T C C G G A G A A G T DNA repair machinery called out: light-dependent repair (accurate) replication block X-rays,  -rays, cosmic rays Generally makes single base-pair changes, or at most very small deletions ---in contrast to:

18 ionizing (higher energy) X-rays,  -rays, cosmic rays generates free radicals:chemical bull in the china shop …among other damage, can induce double-strand DNA breaks cell must repair this damage at any cost to avoid dying after next cell division!! …but what repair template available? If none can be found (or found in time), just stick the DNA together blindly. Hence,ionizing radiation causes just about every genetic change imaginable. (double helix, not sister chromatids)


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