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Announcements1. 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
4. Lab this week: go over quiz and go over more linkage practice problems
5. Practice problems ch. 7: 9, 19.
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?
II. Linkage and mapping in haploid organisms - ordered tetrad analysisD = 1/2(second-division segregant asci)/total
Outline of Lecture 14
I. Somatic cell hybridization - human chromosome maps
II. Overview of Bacterial and Phage Genetics
• Conjugation• Integration• General Recombination• Transformation• Transduction
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.
Example
• Gene A:• Gene B:• Gene C:• Gene D:
Human Chromosome Maps
• 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.
Why didn’t Mendel Observe Linkage?
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
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.
Genetic Recombination Revealed by Selective Media
Colonies ofprototrophs onminimal media
met- bio- thr+ leu+ thi+ met+ bio+ thr- leu- thi-A B
A + B
Cells Must Contact Each Other for Mating: the Davis U tube
How does genetic recombination occur?
Cells that donate = F+ Cells that receive DNA = F-
No growth!
Conjugation: process by which genetic information is transferred, recombined
• Discovered by Lederberg and Tatum (1946)• Genetic info is transferred; basis for mapping
Sex withoutreproduction
Sex pilus is tubethrough whichDNA is passed
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).
(tetracycline, kanamycin, streptomycin, sulfonamide, ampicillin, mercury)
Typical Bacterial Plasmid
Resistance transferfragment
Origin ofReplication
Mechanism of Conjugation: F+ X F-
Pilus often breaks before complete transfer!
two F+ cells result
1 F+ cell 1 F- cell
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???
Mapping by Interrupted Mating in Hfr
• Chromosome transferred linearly
• Gene order and distance between genes could be measured in minutes
Time Map of Experiment
You can infer the orderof the genes on thebacterial chromosome.
“Minutes” = map units
Overlapping Time Maps
The plasmid can insert randomly into the bacterial chromosome,allowing the complete chromosome to be mapped.
F+ to Hfr by Integration into Bacterial Chromosome, Followed by General Recombination
Recombinationlike crossing over
F factor is last to transfer; F- stays F-
Conjugation
F factor integrates
Chromosome transfer
Replication
Circular Map ofE. coli
~2000 genes
Scaled in minutes
One minute = ~ 20%recombination frequency
Transformation: a different process of recombination, can be used to map genes
Bacteriophages are viruses that use bacteria as hosts
Transduction: virus-mediated bacterial DNA transfer
T4 bacteriophage
T4 Phage Self-assembly: Development of a Simple Entity
Head is an Icosahedron (20 faces)
recombinants
Lawn ofbacteria
Larger, darker
Smaller, lighter
Smaller, darkerparental
Larger, lighter
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
T4 Map
rII locus
From Recombination Analysis