Transcript Document

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 inclass 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
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What is the heterozygous arrangement of alleles in
the female parent?
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What is the gene order?
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What are the map distances between each pair of
genes?
II.
Linkage and mapping in haploid organisms - ordered
tetrad analysis
D = 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
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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
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Gene A:
Gene B:
Gene C:
Gene D:
Human Chromosome Maps
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.
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
met- bio- thr+ leu+ thi+
A
B
met+ bio+ thr- leu- thi-
Colonies of
prototrophs on
minimal media
A+B
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!
Conjugation: process by which genetic
information is transferred, recombined
Sex without
reproduction
Sex pilus is tube
through which
DNA is passed
• Discovered by Lederberg and Tatum (1946)
• Genetic info is transferred; basis for mapping
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).
Typical Bacterial Plasmid
(tetracycline, kanamycin, streptomycin, sulfonamide, ampicillin,
mercury)
Origin of
Replication
Resistance transfer
fragment
Mechanism of Conjugation: F+ X F1 F+ cell
1 F- cell
two F+ cells result
Pilus often breaks before complete transfer!
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 order
of the genes on the
bacterial 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
Chromosome transfer
F factor integrates
Conjugation
Replication
Recombination
like crossing over
F factor is last to transfer; F- stays F-
Circular
Map of
E. 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)
Recombination in Phage
Larger, darker recombinants
Lawn of
bacteria
Smaller, lighter
Smaller, darker
parental
Larger, lighter
• 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
rII locus
T4 Map
From Recombination Analysis