Announcements

1. Announcements Lab reports: as printed in the X-linked cross lab write-up, you are expected to perform chi-square analysis on your data (both F1 and F2 for each cross - total of 4 chi-square tests); this will be basis for your discussion, ie. was there significant deviation between expected and observed ratios? New deadline: Fri. Nov. 8th at start of lecture. Problem set 5 is graded and in folders in lab; avg. was 12/15. We’ll go over correct answers in lab this week; we’ll also take questions re. prob. set 6. 3. Ch. 17 rdg: skim pp.456-458; 460-461;

2. Review of Last Lecture • Eukaryotic Gene Expression: it’s more complicated being multicellular 2. The Promoter 3. Enhancers 4. Methylation 5. Alternative splicing

3. Outline of Lecture 28 I. Post-transcriptional gene regulations: Alternative splicing II. Classification of mutations III. Detection of mutations in humans IV. Different forms of mutations

4. I. Post transcriptional gene regulation If humans have approximately the same number of genes as a fruit fly, and we require more complex cellular functions (presumably with a larger number of proteins) - how do we accomplish this? Making different forms of a protein from a single gene Differential splicing of exons

5. Processing Control: Differential Splicing of IgM genes

6. Alternative splicing 1. Chromosomal ratio activates txn of Sxl in females only 2. SXL controls splicing of tra-2 mRNA 3. Females: exon 2 (which has a stop codon) is removed via SXL Males: exon 2 is not removed. Males: no active TRA Females: TRA is made. 5. TRA directs splicing of dsx mRNA in specific manner; in males default splicing occurs.

7. II. Classification of mutations A. Spontaneous vs Induced B. Gametic vs Somatic C. Phenotypic Effect: • Morphological, e.g. pea, fly traits • Biochemical, e.g. hemophilia, leu- • Behavioral, e.g. mating behavior in flies • Regulatory, e.g. in lac operon; important for evolution • Lethal, e.g. Huntington disease, Manx allele • Conditional, e.g. temperature-sensitive allele

8. III. Detection of mutations in humans - Pedigrees - Dominant mutations easiest to detect Where did original mutation occur? Gamete of parent - gen.I Probably not Could it be an X-linked mutation?

9. What is the Rate of Spontaneous Mutation? • Bacteriophage T2: 10-6 to 10-8 per gene replication. • E. coli: 10-5 to 10-10 per cell division. • Maize: 10-5 to 10-6 per gamete per generation. • Drosophila: 10-5 to 10-6 per gamete per generation. • Human: 10-5 to 10-6 per gamete per generation.

10. Neutral vs. deleterious mutations Most spontaneous mutations will occur in 95% of genome that does not encode genes - neutral mutations. What is rate of deleterious mutations in humans? 1.6 deleterious genetic changes/individual/generation Consequences to our species? Read short article by James Crow on page 465-466 in text.

11. IV. Different forms and origins of Mutations

12. More types of mutations • Normal THE ONE BIG FLY HAD ONE RED EYE • Missense THQ ONE BIG FLY HAD ONE RED EYE • Nonsense THE ONE BIG *** • Frameshift THE ONE QBI GFL YHA DON ERE DEY • Deletion THE ONE BIG HAD ONE RED EYE • Insertion THE ONE BIG WET FLY HAD ONE RED EYE • Duplication THE ONE BIG FLY FLY HAD ONE RED EYE • Expanding Mutation • Generation 1 THE ONE BIG FLY HAD ONE RED EYE • Generation 2 THE ONE BIG FLY FLY HAD ONE RED EYE • Generation 3 THE ONE BIG FLY FLY FLY HAD ONE RED EYE

13. Fragile X Syndrome: the Result of an Expanding Mutation Over Time • Mental retardation, characteristic facial structure. • CGG is present in 5’ UTR of FMR-1 gene: • wildtype: 6-54 repeats • carrier: 55-200 repeats • affected: > 200 repeats • > 200 repeats, DNA becomes methylated, inactivating the gene. • Function of protein still unclear. • Other examples: one form of muscular dystrophy, Huntington’s disease.

14. Different forms of mutation 1. Tautomeric Shifts: spontaneous 2. Base Analogues: chemical 3. Alkylating Agents: chemical 4. Intercalating Dyes: chemical 5. Deamination: chemical 6. UV Radiation and Thymine Dimers 7. High-Energy Radiation (X rays, gamma rays, cosmic rays) environmental

15. Tautomeric Shifts Cause Spontaneous Mutations • Can’t be avoided - inherent in the chemistry of bases. • Always a transition: changing one purine (G or A) to the other purine, or one pyrimidine (C or T) to the other pyrimidine. • DNA proofreading minimizes the mutation rate due to tautomeric shifts. This involves 3’-5’ exonuclease activity in DNA polymerase.

16. Keto-Enol Tautomeric Shifts in T and G common rare

17. Amino-imino Tautomeric Shifts in C and A common rare

18. Mispairing Due to Tautomeric Shifts

19. Formation of a TA to CG Transition During DNA Replication Transition is a purine replaced by different purine or pyrimidine replaced by different pyrimidine.

20. 2. Base Analogues: DNA can Incorporate 5-BU in place of Thymine rare common Changes T-A pair > C-G pair. T > C, and A > G are both Transitions

21. 3. Alkylating Agents: Ethylmethane Sulfonate (EMS) Alkylates Guanine Note: changes a G-C pair into an A-T pair (G > A is a transition, C > T is a transition) Another example: mustard gases first used in WWI.

22. 4. Intercalating Dyes Cause Frameshifts Intercalate themselves into the DNA double helix, distorting it, and causing insertion or deletion during DNA replication or recombination. Other examples: Ethidium Bromide, DAPI.

23. 5. Deamination is Caused by Nitrous Acid (a) Causes: C -> T transition (and G -> A transition) (b) Causes: A -> G transition (and T -> C transition). All. Deamination can be spontaneous as well.

24. 6. Ultraviolet Radiation Cause Thymine Dimers Disrupts synthesis; good for sterilization of bacteria, bad for skin cancer. 260 nanometer wavelength

25. 7. High-Energy (Ionizing) Radiation

26. Effects of Ionizing Radiation • Causes either point mutations or breaks in phosphodiester bonds of DNA backbone. • If both strands broken, there can sometimes be repair in mammals through the double-strand break repair (DSB) system. • Dividing cells are more susceptible to therapeutic X-rays than non-dividing cells (radiation therapy for cancer).