Transcript gal - Western Washington University

Seminar
Wednesday, May 15, Biology 212, 4 pm
"Genomic Consequences of Allopolyploidization"
Luca Comai, Ph.D., Department of Botany, University of
Washington
5 points for attendance, 1-5 points each person for good
questions during the Q/A period after the talk.
Mutation in Bacteria
…the ultimate source of variation in
bacteria is spontaneous mutation,
– generally errors in DNA replication,
…mutations occur in specific genes at a rate
of 1 in 106 to 1 in 107 cells,
…adaptive mutations are quickly replicated
and adaptive colonies predominate.
Spontaneous Mutations
• DNA replication in E. coli occurs with an error every ~ 109
bases.
• The E. coli genome is 4.6 x 106 bases.
 An error occurs once per ~ 2000 replications.
• If a single colony has 107 bacteria,
 5,000 cells carry a mutation,
 or, one mutation every ~ 1,000 bases (across a colony),
 or, a mutation in about every gene.
Conjugation
… F+ cells donate genetic material via the F+
plasmid,
…Hfr cells; strains with a chromosome integrated F
factor that is able to mobilize and transfer part of
the chromosome to the F- cell.
’
F Cells
• an F factor from an Hfr cell excises out of the bacterial
genome and returns to plasmid form,
• often carries one or more bacterial genes along,
’
• F cells behave like an F+ cells,
– merizygote: partially diploid for genes copied on the F’plasmid,
• F’plasmids can be easily constructed using molecular
biology techniques.
Selective Media
• wild-type bacteria grow on minimal media,
• media supplemented with selected compounds supports
growth of mutant strains,
– minimal media + leucine supports leu- cells,
– minimal media + leucine + arginine supports leu- arg– etc.
• Selective Media: a media in which only the desired
strain will grow.
Selection
...the process that establishes conditions in
which only the desired mutant will grow.
Problem
Strain
Strain 1
Strain 2
Strain 3
Mating
Type
F'
F'
F-
Chromosome Geno type
F factor Genotype
wild-typ e
argthr- ; thi- ; met-
thr+ ; thi+
thr+ ; thi+
-
You want to create cells that are only methionine auxitrophs.
The Wrong Answer
Strain
Strain 1
Strain 2
Strain 3
Mating
Type
F'
F'
F-
Chromosome Geno type
F factor Genotype
wild-typ e
argthr- ; thi- ; met-
thr+ ; thi+
thr+ ; thi+
-
Strain 1 x Strain 3
You have a recombinant that is met- only.
How do you get rid of Strain 1?
Correct Answer
Strain
Strain 1
Strain 2
Strain 3
Mating
Type
F'
F'
F-
Chromosome Geno type
F factor Genotype
wild-typ e
argthr- ; thi- ; met-
thr+ ; thi+
thr+ ; thi+
-
Strain 2 x Strain 3
Grow on Minimal Media Plus Methionine
Strain 2 dies because there is no arginine.
Strain 3 dies because there is no threonine or thimine.
The new exconjugate lives.
High Frequency of Recombination
(Hfr)
...bacteria exhibiting a high frequency of
recombination,
…the F factor is integrated into the
chromosomal genome.
F factor and Chromosomal DNA
are Transferred
Recombination Requires
Crossing over
Double Crossover
Incomplete Transfer of DNA
• Interrupted Mating: a break in the pilus
during conjugation stops the transfer of
DNA,
• Transfer occurs at a constant rate,
– provides a means to map bacterial genes.
How Do You Interrupt Bacterial
Mating
spread on agar
mate for
specified
time
frappe
Hfr and Mapping
HfrH
strs
thr+
azir
tonr
lac+
gal+
(sensitive to streptomycin)
(able to synthesize the amino acid threonine)
(resistant to sodium azide)
(resistant to bacteriophage T1)
(able to grow with lactose as sole source of carbon)
(able to grow with galactose as sole source of carbon)
Fstrr
thrazis
tons
lacgal-
(resistant to streptomycin
(threonine auxotroph)
(sensitive to sodium azide)
(sensitive to phage T1)
(unable to grow on lactose)
(unable to grow on galactose)
Hfr and Mapping
HfrH
strs
thr+
(sensitive to streptomycin)
(able to synthesize the amino acid threonine)
Fstrr
thr-
(resistant to streptomycin)
(threonine auxotroph)
Streptomycin kills the HfrH cells in the mating mix.
No threonine kills the F- cells in the mating mix.
Hfr and Mapping
HfrH
azir
tonr
lac+
gal+
(resistant to sodium azide)
(resistant to bacteriophage T1)
(able to grow with lactose as sole source of carbon)
(able to grow with galactose as sole source of carbon)
Fazis
tons
lacgal-
(sensitive to sodium azide)
(sensitive to phage T1)
(unable to grow on lactose)
(unable to grow on galactose)
Interrupting Bacterial Mating
spread on selective media
mate 9 min
blend
Replica Plating
After 9 minutes, only azide resistant cells grow.
10 Minutes
Azide, and bacteriophage resistant cells grow.
15 Minutes
Azide, and bacteriophage resistant cells, and lactose utilizing cells.
18 Minutes
All recombinants grow.
Bacterial Map Distances
units = minutes
F factor inserts in different regions of the bacterial
chromosome,
Also inserts in different orientations.
Origin of Replication
Hfr
strain
H
1
2
3
Order of transfer
thr azi ton lac pur gal his gly thi
thr thi gly his gal pur lac ton azi
lac pur gal his gly thi thr azi ton
gal pur lac ton azi thr thi gly his
Indicates direction of transfer.
F factor
A
A
a
Hfr
F-
F factor
A transfers first.
A
A
Hfr
F-
A transfers last.
A
Hfr
A
F-
Leading Gene: the first gene transferred, it is determined empirically.
Hfr
strain
H
1
2
3
Order of transfer
thr azi ton lac pur gal his gly thi
thr thi gly his gal pur lac ton azi
lac pur gal his gly thi thr azi ton
gal pur lac ton azi thr thi gly his
E. coli Map
• 0 minutes is at the
threonine,
• 100 minutes is
required to transfer
complete genome,
Typical Problem
combine
combine
+
Refer to partial maps
for map distances.
11.5 minutes
Join Maps
26 minutes
Practice
• Insights and Solutions, #2,
• Problem 8.17, 8.18, 8.19.
Transformation
• heritable exchange brought about by the
incorporation of exogenous DNA,
– usually DNA from same, or similar species.
Donor and Recipient
Not all cells are competent to receive DNA.
Competence
…a transient state or condition in which a
cell can bind and internalize exogenous
DNA molecules,
…often a result of severe conditions,
– heat/cold,
– starvation, etc.
Competent Cell
Genes are expressed that produce proteins that, in turn, span the cell membrane.
Exogenous DNA Binds Receptor
Complementary Strand Degraded
...one strand of the exogenous DNA is degraded also.
Exogenous DNA Incorporated
Heteroduplex
Cell Divides
Transformation and Mapping
• transformed DNA is generally 10,000 20,000 base pairs in length,
– carries more than one gene,
• When two or more genes are received from
the same transformation event, they are said
to be co-transformed.
Linkage in Bacteria
• genes that are closer together, have a higher
probability of being cotransformed,
– higher probability of being on same donor DNA,
– lower chance of crossover event between genes,
• probability of transformation by two separate
events is low,
• linkage in bacteria refers to proximity.
F factor Review
+
Now F
Now F+
Transduction
…virally mediated gene transfer from one
bacterium to another,
…bacteria viruses are termed bacteriophages.
T4 Bacteriophage
…infects E. coli,
Two Bacteriophage Strategies
• Lytic,
– a type of viral life cycle resulting in the release
of new phages by death and lysis of the host
cell,
• Lysogenic,
– a type of viral life cycle in which the visus
becomes incorporated into the host cell’s
chromosome.
Lytic Cycle
specific transmembrane
phage/bacteria binding
sites,
4. phage reassemble
with repackaged DNA,
1. host cell physiology
is shut down,
virus DNA
inserted into
host cell,
2. host cell physiology
is used for phage work,
3. phage DNA replicated,
capsule parts made,
5. host cell is
degraded and
lyses.
Generalized Transduction
…enzymatic process which can result in the
transfer of any bacterial gene between
related strains of bacteria.
Phage Infects Host
we’ll follow gene C+.
Specific Binding Sites,
Phage DNA inserted,
Upon infection, host cell physiology is shut down,
Phage Hijacks the Host Cell’s
Transcription/Translation
Machinery
gene C+ is present
on a DNA fragment.
Phage replicates own DNA,
makes protein head etc.,
Host cell degraded, the host
chromosome is cut,
Cell Lyses, Phage Move On
C+ is packaged
instead of
phage DNA in
one of
thousands of
new phages,
phage particle
with C+ moves
to another host
cell.
End of the Route
Host Chromosome,
Phage DNA,
packaged
host DNA,
inserted in cell,
inserted in Genome,
via double crossover.
packaged
host DNA,
Virulent Phages
…reproduce via the lytic cycle only.
Two Bacteriophage Strategies
• Lytic,
– a type of viral life cycle resulting in the release
of new phages by death and lysis of the host
cell,
• Lysogenic,
– a type of viral life cycle in which the visus
becomes incorporated into the host cell’s
chromosome.
Lytic vs Lysogenic
viral DNA is
incorporated into
the host genome.
Lysogeny
…the integration of viral DNA into the
bacterial genome,
– a virus that can integrate into the genome
is termed temperate,
– an integrated phage is termed a
prophage.
Prophage
…non-virulent units that are inserted in the
host chromosome, and multiply via binary
fission along with the host DNA,
…prophage can re-enter the lytic cycle to
complete the virus life cycle.
Phage Induction
…prophage express a repressor protein that inhibits
further infection,
– also inhibits prophage DNA excision genes, and genes
used during the lytic cycle,
…environmental cues (especially events that damage
DNA) block the expression of the repressor
protein,
– prophage excises and enters a lytic cycle.
Specialized Transduction
…upon excision of the prophage, adjacent
host DNA is taken along,
…the completion of the lytic cycle and
subsequent infection of another host moves
the flanking DNA to another bacterium.
Normal Excision
Abnormal Excision
flanking DNA is removed.
Transfer to Other Cells
Biotechnology
• Bacteria: again with the gene therapy,
– here they are harnessing another organism to do
their dirty work,
• Humans: use phages to do a variety of
molecular biology work,
– also use virus to deliver genes in new gene
therapy technologies.
T Plasmids
…bacteria also have plasmids that they transfer to
other organisms,
…upon infection, the T plasmid enters the host cell,
becomes incorporated in the host genome, and the
T plasmid genes become expressed,
…Agrobacterium tumefaceins transfers genes that
force plants to make strange sugars, that only the
Agrobacterium can digest.
Transposable Elements
…a segment of DNA that can move to, or move a
copy of itself to another locus on the same or a
different chromosome (hopping DNA),
…may be a single insertion sequence, or a more
complex structure (transposon) consisting of two
insertion sequences and one or more intervening
genes.
Bacteria are Geniuses
• Cloning: identical copies,
• Gene therapy: insertion of a healthy, or functional
gene into a organism lacking a good gene,
• Defense: develop genes to ward off poisons,
predators, etc.
• Genetic engineering: inserting DNA into another
organism to do your bidding,
• Harness Mutation: to speed evolution.
Weds.
• Work the Assigned Problems.
• Study the Benzer Experiment.