Announcements

1. Announcements • 1. Survey results: 87% like powerpoint • 85% print notes before class • 93% thought exam 1 covered appropriate material • 43% thought exam 1 was appropriate length • Suggestions I will consider: posting lecture notes earlier, making exam 2 a bit shorter, more practice problems, continue doing problems during lecture. 2. Consider whether you prefer class to meet Wed. and not Fri., and no in-class review on Wed. before exam 2 OR in-class review Wed. and class meets Friday (day of exam). We’ll vote Friday. 3. Average on quiz 2 = 6.83/12 • Lab this week: go over quiz and go over more linkage practice problems 5. Practice problems ch. 7: 9, 19.

2. Review of Last Lecture I. Determining the order of genes, continued - example in maize • What is the heterozygous arrangement of alleles in the female parent? • What is the gene order? • What are the map distances between each pair of genes? • Linkage and mapping in haploid organisms - ordered tetrad analysis D = 1/2(second-division segregant asci)/total

3. Outline of Lecture 14 I. Somatic cell hybridization - human chromosome maps II. Overview of Bacterial and Phage Genetics • Conjugation • Integration • General Recombination • Transformation • Transduction

4. I. Human Chromosomes have been Mapped by Somatic-cell Hybridization • Two cells from mouse and human fused to form heterokaryon (two nuclei in common cytoplasm). • Nuclei fuse to form synkaryon and lose human chromosomes over time. • Gene products are assayed and correlated with remaining human chromosomes. • Genes also mapped by pedigree analysis and recombinant DNA techniques.

5. Example • Gene A: • Gene B: • Gene C: • Gene D:

6. Human Chromosome Maps

7. Why didn’t Mendel Observe Linkage? • There are 7 chromosomes and 7 genes • Did he get one gene per chromosome? • Genes are located on four chromosomes, but far enough apart to seem unlinked (frequent crossing over creates independent assortment). • He should have seen linkage if he had mated dwarf plants with wrinkled pea, but he apparently didn’t do this experiment.

8. II. Escherichia coli • A model organism: useful for discovering general principles common to all organisms. • The focus of genetic research from the 1940’s to 1960’s: What is a gene and how does it work? • Advantages: short doubling time (30 min), simple culture media, pure cultures, haploid, lots of mutations. • The advantage of being haploid is that a mutation in the parent is always seen in the offspring. • In diploid organisms, mutations can be covered up if they are recessive. • Bacteria are haploid • Sordaria are haploid

9. Growth • E. coli can grow on carbon source (e.g. glucose) + minimal inorganic salts. • Prototrophs: Grow well, are wildtype. • Auxotrophs: Require some other organic molecule that it cannot make, due to a mutation (e.g. amino acid leucine - leu-). • Grow in liquid culture flask or petri dish.

10. Genetic Recombination Revealed by Selective Media met+ bio+ thr- leu- thi- met- bio- thr+ leu+ thi+ A B Colonies of prototrophs on minimal media A + B

11. How does genetic recombination occur? Cells Must Contact Each Other for Mating: the Davis U tube Cells that donate = F+ Cells that receive DNA = F- No growth!

12. Conjugation: process by which genetic information is transferred, recombined Sex pilus is tube through which DNA is passed Sex without reproduction • Discovered by Lederberg and Tatum (1946) • Genetic info is transferred; basis for mapping

13. Requirements for conjugation: F+ X F- Bacteria • Two mating types exist: donor F+ (fertility) cells and recipient F- cells. • Physical contact through F pilus on F+ cells is required for conjugation. • F+ cells contain a fertility factor (F factor): - any cells grown with F+ become F+, F factor appears to be a mobile element - a plasmid (circular, extrachromosomal DNA) containing: (1) genes to allow transfer of plasmid (RTF) and (2) antibiotic resistance genes (r-determinants).

14. Typical Bacterial Plasmid (tetracycline, kanamycin, streptomycin, sulfonamide, ampicillin, mercury) Origin of Replication Resistance transfer fragment

15. Mechanism of Conjugation: F+ X F- 1 F+ cell 1 F- cell two F+ cells result Pilus often breaks before complete transfer!

16. Hfr bacteria and chromosome mapping Hfr = high frequency of recombination This is a special type of F+, acts as donor of chromosome F+ x F- F+ Hfr x F- F- Some genes recombined more often than others???

17. Mapping by Interrupted Mating in Hfr • Chromosome transferred linearly • Gene order and distance between genes could be measured in minutes

18. Time Map of Experiment You can infer the order of the genes on the bacterial chromosome. “Minutes” = map units

19. Overlapping Time Maps The plasmid can insert randomly into the bacterial chromosome, allowing the complete chromosome to be mapped.

20. F+ to Hfr by Integration into Bacterial Chromosome, Followed by General Recombination Chromosome transfer Replication F factor integrates Recombination like crossing over Conjugation F factor is last to transfer; F- stays F-

21. Circular Map ofE. coli ~2000 genes Scaled in minutes One minute = ~ 20% recombination frequency

22. Transformation: a different process of recombination, can be used to map genes

23. Bacteriophages are viruses that use bacteria as hosts

24. Transduction: virus-mediated bacterial DNA transfer

25. T4 bacteriophage

26. T4 Phage Self-assembly: Development of a Simple Entity Head is an Icosahedron (20 faces)

27. Recombination in Phage • Strains with different plaque morphologies “crossed” by coinfection of bacteria: h r+ X h+ r • h mutant plaques are darker than h+ • r mutant plaques are larger than r+ • Results: parental (h r+ and h+ r) and recombinant (h+ r + and h r) plaques. • # recombinants/total X 100% = recomb. frequency Larger, darker recombinants Lawn of bacteria Smaller, lighter Smaller, darker parental Larger, lighter

28. rII locus T4 Map From Recombination Analysis