Download repair - Molecular and Cell Biology

Terminology
for chromosomal
rearrangements:
Df = deficiency = deletion
(used in connection with
Muller's mutant allele
category tests to manipulate
gene dose)
(used to reduce allele dose)
A physical map
of the genome
in Muller's day
Bulge in the synapsed
polytene chromosomes
of a heterozygote
shows what is deleted
Fig. 14.8
Terminology
for chromosomal
rearrangements:
(used in connection with
Muller's mutant allele
category tests to manipulate
gene dose)
Df = deficiency = deletion (used to reduce allele dose)
Dp = duplication (insertional vs. tandem)
(used to increase allele dose)
Bulge in the synapsed
polytene chromosomes
of a heterozygote
shows what is deleted
(or duplicated)
Such rearrangements
helped workers locate
genes on the polytene
chromosome map.
Inversions also have helped locate genes on the Drosophila
polytene chromosome map
(In)
Inversion breakpoints
Drosophila
life cycle
@25C
adult
(reproductive in
~2 days)
4 days
fertilized egg
(embryo)
Pupa
(metamorphosis)
¢
™
XY
XX
1 day
1st instar larva
1 day
2 days
2nd instar larva
3rd instar larva
1 day
If a gene functions at different times to do different things,
temperature shifts of ts mutants
can reveal those various functions
ultimate phenotype
determination of the “temperature-sensitive period” (TSP)
by single temperature shifts up or down
permissive
unshifted
all OK
shift rstrctv
to prmsv
TSP start:
when begins drop
TSP
TSP end:
when wildtype
shift prmsv
to restrv
all mut
embryo
larva
pupa
adult
restrictive
unshifted
point (time) in development when temp. shifted
determination of the “temperature-sensitive period” (TSP)
ultimate phenotype
Temperature pulses (double-shifts) are more definative:
R
shift from prmsv
to restrv AND BACK
P P
gene function needed:
1
2
all OK
permissive
unshifted
TSP
restrictive
unshifted
all mut
embryo
larva
pupa
adult
point (time) in development when exposed to rstrctv. temp for
some fixed period of time (relatively short for high resolution).
Temperature-sensitive mutant alleles help us recognize
(“dissect”) a gene’s pleiotropy (multiple, “unrelated” functions)
ultimate phenotype
time of function
essential for
Consider if: development
two TSPs:
all OK
viability
vs.behavior
&
1
time of function
needed for an
adult behavior mutant animal survives,
allowing us to see
the behavioral abnormality
2
permissive
unshifted
viability
low
behavior
abnormal
restrictive
unshifted
all mut
embryo
larva
pupa
adult
point (time) in development when exposed to the restrictive temp.
Mutations (changes in DNA):
the lifeblood of genetic analysis
(1) What kinds can we make? (categories)
(2) How do we make them? (mutagenesis)
(3) How do we find them? (mutant screens & selections)
(4) Why bother?
Factors affecting Mutation Rates
gamete
MEASURE: changes per gene per: cell division
generation
Spontaneous mutations:
DNA mistakes happen (nothing is perfect)
Why do they occur at the rate they do?
Consider:
(1) It costs energy and time to avoid making mistakes
…proofreading of DNA replication
DNA synthesis and repair genes
determine the spontaneous mutation rate
“mutator” mutations: increase spontaneous rate
(2) It doesn’t pay to be perfect
Natural selection optimizes spontaneous mutation rate
redwood trees vs. E.coli
Mutagens: agents that increase mutation rate
Chemicals:
(Fig. 7-10, learn for MCATs)
(1) base analogs
incorporated in place of normal, then misbehave
5-BrU (5-bromouracil) incorp like T , generally pairs w/ A),
but tautomerizes to pair with G
(2) base modifying agents
modifications change pairing rules
EMS (ethylmethane sulfonate)
Ethylated G pairs with T
Ethylated T pairs with G
induces replication error
GC >GC>G*>GT>*T >AT
TA>CG
induced transition or transversion?
(3) intercalating agents
slip in between bases noncovalently
cause additions and deletions
acridine orange
Mutagens: agents that increase mutation rate
Chemicals:
(1) base analogs
(2) base modifying agents
(3) intercalating agents
(4) original root beer (sasparilla root)
organic all-natural carcinogen
(5) urine from smokers
Ames test for mutagens [p224. Fig 7.16]
Salmonella typhimurium
used a panel
of different
molecularly
defined mutants
histidinehistidine+ reversion rate
requires
does not require
auxotroph
prototroph
With chemical mutagens:
(1) …generally easier to induce germline mutations
in males than in females.
in sperm: little to “distract” the mutagen from DNA
With chemical mutagens:
(2) …often progeny from mutated sperm are
genetically mosaic for new mutations
only one of the two complementary bases in sperm
are modified: GC ->
G *C
-> G*T & GC
in sperm
after 1st zygotic division
subsequent rounds of replication
do not invariably cause errors:
Question: if an animal is mosaic
for the new mutant base pair, under what
(developmental) circumstances will that new mutation
not be transmitted to the progeny?
(wt)
G*T -> G*C & AT
wt
mutant
Answer: if the mutant base pair doen't end up in germline stem cells.
Radiation (the first experimental mutagen discovered)
non-ionizing (lower energy)
UV light
photochemical reaction glues adjacent thymines together in cis:
replication block
A G G C C T C TT T C A
A G G C C T C T=T C A
T C C G GA GAA G T
TCCGGAGA AGT
DNA repair machinery called out:
light-dependent repair (accurate)
excision repair (error prone)
(pity the poor shoe salesmen of yore)
ionizing (higher energy)
X-rays, -rays, cosmic rays
Radiation (the first experimental mutagen discovered)
ionizing (higher energy)
X-rays, -rays, cosmic rays
generates free radicals: chemical bull in the china shop
…can induce double-strand DNA breaks
important to repair at any cost!!
…but what repair template available?
(double helix shown, not sister chromatids)
-- information on the sister chromatid, but only after DNA replication
-- information on the homologous chromosome in diploid organisms
If none can be found (or found in time),
just stick the DNA together blindly.
Hence,ionizing radiation causes just about every
molecular category of genetic change imaginable.
Reading:
“Transposable genetic elements move
from place to place in the genome”
pp508-514 (Chp 14)
Also: p548 (insertion sequences in bacteria)
A practice problem set dealing with mutant categories
is available on the website.
Answers will be posted next Monday.
Lecture schedule:
Wed 3/14: genetic screens
Fri 3/16: sensitized screens
Mon 3/19: genetic mosaics in screens
(hence: no RNAi and one less sex lecture)
Radiation (the first experimental mutagen discovered)
non-ionizing (lower energy)
UV light
photochemical reaction glues adjacent thymines together in cis:
A G G C C T C TT T C A
T C C G GA GAA G T
replication block
A G G C C T C T=T C A
TCCGGAGA AGT
DNA repair machinery called out:
light-dependent repair (accurate)
excision repair (error prone)
Generally makes single base-pair changes,
or at most very small deletions ---in contrast to:
ionizing (higher energy)
X-rays, -rays, cosmic rays
ionizing (higher energy)
X-rays, -rays, cosmic rays
generates free radicals: chemical bull in the china shop
…among other damage, can induce double-strand DNA breaks
(double helix, not sister chromatids)
cell must repair this damage at any cost
to avoid dying
after next cell division!!
…but what repair template available?
If none can be found (or found in time),
just stick the DNA together blindly.
Hence,ionizing radiation causes
just about every genetic change imaginable.