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