Genetics

Stochastic Effects:Genetic Effects of Ionizing Radiation
Stochastic
Genetic effect
Effects
Introduction
Structural changes
genetic effects of
Chromosome breakage ionizing radiation
Breakage hypotheses
by: AR Mahdizadeh
Gene mutations
Muller’s Sex-linked test
Specific Locus test(mice) From: Radiation Biophysics
Edward L. Alpen
Chapter 13 pages:283-299
Gene mutation in cultured mammalian cells
Introduction
Genetic effect • The change in DNA of the
Introduction gametes (Genetic Mutation)are
Structural changes indeed of the stochastic class.
Chromosome breakage
• They occur on probabilistic basis
Breakage hypotheses
with no threshold for the
Gene mutations occurrence and the severity of the
Specific Locus test(mice)
phenotypic changes resulting from
them is unrelated to dose.
RADIATION BIOPHYSICS.1990 E. ALPEN 2

Introduction
Genetic effect • Loss of clonogenic potential is due
Introduction to defect in the DNA of the cell
Structural changes • The same mechanism will also
Chromosome breakage
lead to possible loss of ,or
Breakage hypotheses
alteration in, the genomic info.
Gene mutations
• If the genomic change exist in the
Specific Locus test(mice) gametes ,it may be passed on to
future generations at the time of
reproductive activity.
Introduction
Genetic effect • Genetic mutation
Introduction →Somatic mutation
Structural changes
→Not transmissible to offspring
Chromosome breakage
Breakage hypotheses →Altered function or phenotype
Gene mutations →An important class is CANCER
• At that time only we could study
metaphase chromosomes.then the
changecells
Gene mutation in cultured mammalian classified in two class
according to visibility .
Introduction
Genetic effect
Introduction
Structural changes • Either gamete or somatic dividing
Chromosome breakage cell,to express the changes that
Breakage hypotheses
have occurred in its genome must
Gene mutations be capable of division.

Structural changes in
chromosome
Genetic effect • Earliest work on chromosome changes
was carried out on the salivary gland of
Introduction the fruit fly
Structural changes
• All nonviable chromosome form
Chromosome breakage
produced in the irradiated sperm will
Breakage hypotheses have been eliminated in the course of
Gene mutations production of larva that is examined.
Muller’s Sex-linked test • Bender and Gooch(1962)
Specific Locus test(mice) • Development of the techniques to
examine the post irradiation
Gene mutation in cultured mammalian cells of mammalian and indeed
chromosome
human lymphocyte.
Structural changes in
chromosome
Genetic effect • Metaphase chromosome represents
Introduction only a brief portion of the life
Structural changes cycle of the chromosome.
Chromosome breakage
• During this “invisible”period
Breakage hypotheses
much of importance is happening.
Gene mutations
• Waldren and Johnson(1974)
Specific Locus test(mice) • New techniques developed for the
examination
of interphase
chromosome.
Chromosome breakage
Genetic effect • Ionizing radiations is the most
Introduction effective cause in producing this
Structural changes class of structural change.
Chromosome breakage • A little consequence as a result of radiation damage is
Breakage hypotheses
maldistribution of chromosomes.
(Daughter cells have an incorrect complement of intact chromosome)
Gene mutations  Breakage before DNA replication

Muller’s Sex-linked test The structural defect will also be replicated
Specific Locus test(mice) (or it will failed the attempted replication)
 Breakage after DNA replication
Asymmetric change in one of the two chromatids.

Chromosome breakage
Genetic effect • Time from the irradiation event
Introduction to the collection of mitosis will
Structural changes determine the position of the cell
Chromosome breakage cycle that is being observed.
Breakage hypotheses
• Three types:
Gene mutations
• Subchromatid (prophase-hard to
Specific Locus test(mice) distinguish)
• Chromatid (post replication G2)
• Chromosome (pre synthesis G1)
Chromosome breakage
Genetic effect • Waldern and Johnson(1974)
Introduction • The majority of the breaks will be
Structural changes restored to their normal
Chromosome breakage condition(restitution)
Breakage hypotheses • Rejoining to a site that is incorrect
Gene mutations causing a transposition of
Muller’s Sex-linked test nucleotide
• Dalrymple(1973)
Alphabetic marker indicates position on
unreplicated chromatide.

Single hit breakage

Genetic effect
Introduction
Structural changes
Chromosome breakage
Breakage hypotheses
A B C.D E F G H I
Gene mutations
A B C .D E F and G H I

Inversion of the fragment or
rejoining at the wrong end
Genetic effect • A B C.D E F I H G
Introduction • I H G A B C .D E F
Structural changes
• G H I A B C .D E F
Chromosome breakage
Breakage hypotheses
Gene mutations

Two-hit breakage
Genetic effect
Introduction
A B C.D E F G H I
Structural changes
Chromosome breakage
Breakage hypotheses
A B C.D, E F and G H I
Gene mutations
Muller’s Sex-linked test A B C.D G H I
Specific Locus test(mice) Interstitial deletion and EF is a minute
Gene mutation in cultured mammalian cells E F A B C .D I H G
Inversion reordering without loss
Multiple hits in replicated
chromosome
Genetic effect • Exchange of parts of the DNA between
the two chromatids or chromosome is a
Introduction possibility .
Structural changes
Chromosome breakage
Breakage hypotheses Isocentric break
Gene mutations

Multiple hits in replicated
chromosome
Genetic effect • Dicentric product
• Movement of parts is called “translocation”
Introduction • If all genetic info. Is intact it called”balanced
translocation”.
Structural changes • Chemical damage leads to “sister chromatid
Chromosome breakage exchange”
Breakage hypotheses Dicentric chromosome
Gene mutations

Breakage hypothesis
Genetic effect • Sax (1940)
Introduction • Lea’s target theory:
Structural changes  Aberrations that requires one break(hit)should
Chromosome breakage
follow the dose response relationship that is
first order in dose.
Breakage hypotheses
 Aberrations that requires two or three
Gene mutations breaks(hits)should follow the dose response
Muller’s Sex-linked test relationship that is respectively second or third
Specific Locus test(mice) order in dose.
Target model has not entirely explain the
data for chromosome aberration.

Breakage hypothesis
Genetic effect • Revell(1955)
Introduction • “achromatic gap”non-staining
Structural changes region of the chromatid the D-R
Chromosome breakage curve was first order in dose.
Breakage hypotheses • On the other hand the frequency of
Gene mutations true chromatid deletions,which
Muller’s Sex-linked test should follow a first-order
Specific Locus test(mice) relationship,were best described
by the dose to the 1.71 power.
• “Exchange hypothesis”
Exchange hypothesis
Genetic effect • All chromosome aberrations arise
Introduction
from an exchange process that has
Structural changes
much in common with,and many
Chromosome breakage
be similar to,meiotic crossing over
Breakage hypotheses that occurs normally during the
division stage of the gamete.
Gene mutations
• The production of chromosome
Specific Locus test(mice) aberration is very complicated.
• The simple breakage model must
,to great extent, be put aside.
Gene mutation
Genetic effect • The structural change in chromosome
are relatively gross disturbances in the
Introduction genetic material of the cell,they often
Structural changes lead to such metabolic and functional
Chromosome breakage disturbances in the cell that no or only a
Breakage hypotheses few division will occur before death.
Gene mutations • Many more subtle changes occur that
Muller’s Sex-linked test are not visible in the morphology but
Specific Locus test(mice) their presence known through alteration
of some genetically controlled function
of the cell.these
Gene mutation in cultured mammalian cells are
”gene mutation”
Gene mutation
Genetic effect • The small changes are called”point
mutations” they give raised to a change
Introduction in a codon that will miss read for a
Structural changes single amino acid.
Chromosome breakage
• Second important type of mutation can
Breakage hypotheses arise as the result of minor
Gene mutations deletion,addition, or substitution
Muller’s Sex-linked test called”frame shift”.
Specific Locus test(mice) Using nonsense codon CAT.
• CAT CAT CAT CAT CAT CAT...
Gene mutation in cultured mammalian cells CA Deleted
• CAT CAT CAT CAT TCA TCA...
Gene mutation
Genetic effect • In the absence of radiation ,the
Introduction mutation rate for a single gene
Structural changes may be on the order of one
Chromosome breakage mutation or less per gene site per
Breakage hypotheses
million cells that are formed
Gene mutations • Radiation can easily raise this rate
by a thousand fold or more.

Gene mutation
Genetic effect • DeSerres,Milligan, and Wbber(1967)
• They studied two adjacent gene
Introduction
producing Adenine.
Structural changes
Chromosome breakage
Breakage hypotheses Yield=0.3+5.39D+0.16 D²
(mutation per million surviving cells),D in kilorad=10Gy
Gene mutations
Muller’s Sex-linked test • Point mutation required one hit and
Specific Locus test(mice) chromosome deletion required two hits.
• This formula provide interesting insight
to L-Q molecular model

Muller’s sex-linked recessive test

Genetic effect • Muller(1927)
Introduction • Developed truly elegant test
Structural changes system to reveal the presence of
Chromosome breakage
lethal mutation anywhere on the X
Breakage hypotheses
chromosome of the fruit
Gene mutations fly,Drosophila melanogaster.
Specific Locus test(mice) • For being the first to show that
radiation could produce mutation
events.

Genetic effect • Muller(1927)
Introduction
Structural changes
Chromosome breakage
Breakage hypotheses
Gene mutations
Nobel Prize


Genetic effect • X/X female fly
Introduction • C only procedural interest
Structural changes
• l lethal gene
Chromosome breakage
Breakage hypotheses • B morphological marker
C Suppresses the crossing over
Gene mutations B Bar-eyed to identify flies that carry the l gene
 C l B/+ (+ is sufficient, if present,to
prevent the lethal but recessive action
of l gene)


Genetic effect • Cross the C l B/+ female with wild
type males that have been irradiated at
Introduction
dose chosen by the investigator.
Structural changes
Chromosome breakage
• The competent, wild-type male is
Breakage hypotheses coded +/Y to indicate that it also has
an X chromosome (+), which will
Gene mutations
prevent the action of l.
Specific Locus test(mice) • Not suffered a radiation induced
mutation in the X chromosome:
Gene mutation in cultured
4) mammalian cells
bar-eyed daughters C l B/+
5) +/+ female will not bar-eyed and excluded


Genetic effect C l B/+ ClB +
+/Y
Introduction +(lm) C l B/+(lm)  +/+ 
Structural changes Bar-eyed not bar-eyed
Chromosome breakage Y Y/C l B Y/+

not bar-eyed
Breakage hypotheses dead
Gene mutations
C l B/+(lm) ClB +(lm)
+/Y
+ C l B/+  +/+ 
not bar-eyed
Bar-eyed
Gene mutation in cultured mammalian
Y cells Y/C l B Y/+(lm)
dead dead


Genetic effect • No male survive from this cross
Introduction (table 2).
Structural changes • The lack of surviving males from
Chromosome breakage
the cross is the sign that a
Breakage hypotheses
mutation occurred in the irradiated
Gene mutations male grandparent,and the number
of such crosses lacking male
survivors is the number of original
irradiated
Gene mutation in cultured mammalian cells males with mutations.


Genetic effect • this elegant experience allowed
Introduction Muller to construct dose-effect
Structural changes curves for this mutation in D.M.
Chromosome breakage
• Muller recessive lethal model
Breakage hypotheses
doesn’t permit us to examine the
Gene mutations sensitivity of female for radiations
mutogenesis.
• The conclusion is as Follow

Conclusion Muller’s sex-linked recessive test

Genetic effect 1. From 25-4000 rad the rate of
production of sex-linked recessive
Introduction
lethal is constant per unit dose.
Structural changes
Chromosome breakage 2. There appears to be no threshold
Breakage hypotheses below which an effect would not been
predicted.
Gene mutations
Muller’s Sex-linked test 3. There is no effect of dose rate over a
Specific Locus test(mice) wide range .there is also no effect of
fractionation of the dose .


Genetic effect
1. The sensitivity of the male
Introduction gamete varies greatly with the
Structural changes stage of development at which it
Chromosome breakage is irradiated
Breakage hypotheses
Gene mutations


Genetic effect
5.The relative sensitivity of the
Introduction female gamete has been measured
Structural changes by another method (specific
Chromosome breakage locus)the value are as follow:
Breakage hypotheses
oocytes and oogonia 1
Gene mutations
late oocytes 2
Specific Locus test(mice) 6.The induction of mutation by
inoizing radiation
in DM will
occur at the rate of 1.5-8 X 10^-8
Specific locus test in mice

Genetic effect • Concern about the reliability of
Introduction Drosophila data for projections to
Structural changes human beings led to the
Chromosome breakage establishment in the early 1960s of
Breakage hypotheses
a large project as Oak Ridge
Gene mutations National Laboratory (ORNL) to
establish the sensitivity of a
mammalian species for radiation
mutagenesis
Gene mutation in cultured mammalian cells .


Genetic effect • Russell’s experimental mouse colony in
which nine mouse specific locus end
Introduction points were available for them to study.
Structural changes
• Great majority of mutations produced
Chromosome breakage
will be recessive.
Breakage hypotheses
• The”specific locus” method is suitable
Gene mutations for this purpose.
Muller’s Sex-linked test • Specific phenotypic expressions are
Specific Locus test(mice) chosen that can be easily detected in the
offspring.like”curly wing”in Drosophila
or ”kinkycells
tail” in mouse.

Genetic effect
Introduction
Structural changes
Chromosome breakage
Breakage hypotheses
Gene mutations

Hope is eternal

Genetic effect
Introduction
Structural changes
Chromosome breakage
Breakage hypotheses
Gene mutations

The longer we wait before the mating,the earlier will be the stage
of maturation of the gamete

Genetic effect  For the seven of loci studied in the
Introduction mouse the average mutation rate per
Structural changes locus ,per rad(per cGy) was found to
Chromosome breakage be 2.2 X 10^-7
Breakage hypotheses
 Study with male mice were
Gene mutations undertaken at dose rate from 0.009-
90 rad/min showed repair process
dose-rate,not dose,dependent and
Gene mutation in cultured saturated at 0.009 rad/min dose rate.
mammalian cells

Specific locus test in mice(male)

Genetic effect
Introduction
Structural changes
Chromosome breakage
Breakage hypotheses
Gene mutations

Specific locus test in mice(female)

Genetic effect
Introduction
Structural changes
Chromosome breakage
Breakage hypotheses
Gene mutations

Mutation in Cultured mammalian cell

Genetic effect Deserres, Malling, and Webber(1967)
• Mutation rates in fungal organism.
Introduction
Structural changes
• In the last two decades similar studies
Chromosome breakage
conduct for mammalian cells .(not for
mammalian gamete but for somatic
Breakage hypotheses
cells)in three types:
Gene mutations 4) Auxotrophic mutants
Muller’s Sex-linked test 5) Temperature sensitive mutants
6) Certain drug resistant
Mutation frequencies for cultured

somaticcells
Gene mutation in cultured mammalian mammalian cells are essentially
identical to mouse specific locus
mutation frequency (1.5-3 X 10^-7)
Genetic effect
Introduction
Structural changes
DNA Strand Breaks

Chromosome breakage
Breakage hypotheses
Gene mutations

DNA Strand Break

Genetic effect
• DNA is the principal target for the
Introduction
biological effects of radiation
Structural changes
Chromosome breakage
– Cell killing
Breakage hypotheses – Mutation
Gene mutations – Carcinogenesis
• Result breaks in DNA, caused by:
– Charged-particle tracks
– Chemical
Gene mutation in cultured mammalian cells species produced

DNA Strand Breaks

Genetic effect • DNA
– Deoxyribonucleic acid
Introduction
– Double helix structure
Structural changes
• 2 strands → held together by hydrogen bone
Chromosome breakage
between the bases
Breakage hypotheses • “Backbone” of each strand → alternating sugar
+ phosphate group
Gene mutations
• 4 bases attached to the “backbone”
– The sequence of the bases → specifies the
Specific Locus test(mice) genetic code
– Single ring group → Pyrimidines (e.g. thymine
& cytosine)
– Double ring group → Purines (e.g. adenine &
guanine)

structure The of a single strand of DNA
Genetic effect
Introduction
Structural changes
Chromosome breakage
Breakage hypotheses
Gene mutations
The structure of a single strand of DNA

DNA Strand Break

Genetic effect • DNA (Con’t)
– Base on the opposite strands must be
Introduction
complementary
Structural changes
– Adenine (A)  Thymine (T)
Chromosome breakage
– Guanine (G)  Cytosine (C)
Breakage hypotheses
Gene mutations

DNA Strand Breaks

Genetic effect • The cell are irradiated with x-rays
– Many breaks of a single strand
Introduction • Little biological consequence
Structural changes • Repair readily using the opposite strand as a template
• Mutation occurred if the repair is incorrect
Chromosome breakage
Breakage hypotheses
Gene mutations
Muller’s Sex-linked test –Both strands are broken, breaks are well separated
Specific Locus test(mice) •Also repair readily
•Two breaks are handled separately

DNA Strand Breaks

Genetic effect • The cell are irradiated with x-rays
(Con’t)
Introduction – Breaks in the two strands
Structural changes • Opposite one another
Chromosome breakage • Separated by only a few base pairs
Breakage hypotheses • Cause double-strand break
– Chromatin snaps into two pieces
Gene mutations – Most important lesion by radiation
– Result in cell killing, mutation, or
carcinogenesis

DNA Strand Breaks

Genetic effect • Double-strand breaks
Introduction – Types:
Structural changes • Distance between the breaks on the two
Chromosome breakage DNA strands
Breakage hypotheses • Kinds of end groups formed
Gene mutations – Yield: 0.04 x of single-strand breaks

Muller’s Sex-linked test – Induced linearly with dose
Specific Locus test(mice) • Formed by single tracks of ionizing
radiation

DNA Strand Breaks

Genetic effect – Repair
• Homologous recombination
Introduction – Requiring an undamaged
Structural changes DNA strand as participant in
the repair
Chromosome breakage – End-to end rejoining via
Breakage hypotheses nonhomologous
recombination
Gene mutations – Error free process
Muller’s Sex-linked test – Rare in mammalian cells
– carried out by proteins similar
to rad51 gene (product of
yeast S.cerevisiae)

DNA Strand Breaks

Genetic effect – Repair
• Nonhomologous recombination
Introduction – Illegitimate recombination
Structural changes – Account for many of the
premutagenic lesions induced
Chromosome breakage in the DNA of human cells by
Breakage hypotheses ionizing radiation
– Participated protein
Gene mutations » DNA-dependent kinase
Muller’s Sex-linked test » Ku protein
Specific Locus test(mice) – Protein complex (recent
research)
» hMre11, Hrad50
Gene mutation in cultured mammalian cells(the product of the
» p95
NBS1 gene)

DNA Strand Breaks

Genetic effect • Double-strand breaks (Con’t)
Introduction – Ionizing radiation
Structural changes • Energy is not deposited uniformly in
Chromosome breakage the absorbing medium
Breakage hypotheses • Located along the tracks of the charged
particles
Gene mutations – Electrons → x-rays or γ-rays
– α-particles → neutrons
• “Spur”, “Blobs” and “short tracks”

DNA Strand Breaks

Genetic effect • Ionizing radiation (Con’t)
– “Spur”
• Contain up to 100 eV
Introduction • Involved 3 ion pairs
Structural changes • 95% of energy deposition by x- or γ-rays
• Diameter: 4 nm (2x diameter of the DNA double helix)
Chromosome breakage – “Blobs”
Breakage hypotheses • Less frequent for x- or γ-rays
• Frequent in densely ionizing radiation (e.g. neutrons, α-
particle)
Gene mutations • Diameter: 7 nm
Muller’s Sex-linked test • Involved 12 ion pairs
Specific Locus test(mice) • More difficult for the cell repair
– “Spurs” & “blobs” have dimension similar to DNA double
helix
Gene mutation in cultured mammalian cells radical attack occurs if they overlap the DNA
• → multiple
helix.
• Wide variety of complex lesions
• Base damage
• Double strand breaks
Genetic effect
Introduction
Structural changes
Chromosome breakage
Breakage hypotheses
Gene mutations
Illustration of a locally multiply damage site
• Locallycells
Gene mutation in cultured mammalian multiply damaged site
– Multiple damage spread out up to 20
base pairs
Genetic effect
Introduction
Structural changes
Chromosome breakage Measuring
Breakage hypotheses
DNA Strand Breaks
Gene mutations

Measuring DNA Strand Breaks

Genetic effect • Isolating the DNA from irradiated cell
– DNA pieces to pass through a porous
Introduction
substrate (e.g. gel, filter)
Structural changes
– DNA pieces move under the influence of
Chromosome breakage
• Gel → electric field
Breakage hypotheses
• Filter → flow
Gene mutations • Small pieces → faster & farther
Muller’s Sex-linked test • Large pieces → slower & nearer
Specific Locus test(mice) – Measuring single strand breaks → strong
alkaline preparation
– Measuring
Gene mutation in cultured mammalian cells double strand breaks → neutral
preparation

Genetic effect • DNA in cell is more resistant to damage
by radiation (c.f. free DNA)
Introduction
– Presence of low molecular weight
Structural changes
scavenger in cells
Chromosome breakage
• Mop up some of the free radicals
Breakage hypotheses
– Physical protection by packaging
Gene mutations • Sequence-specific sensitivity
Muller’s Sex-linked test • Actively translating genes → more sensitive to
Specific Locus test(mice) radiation


Genetic effect • D0 dose
– One lethal event / cell
Introduction – 37 % still viable
Structural changes
– e.g. mammalian cell → D0 lies between 1
Chromosome breakage – 2 Gy
Breakage hypotheses • Number of DNA lesions per cell
– Base damage > 1000
Gene mutations – Single-strand breaks about 1000
Muller’s Sex-linked test – Double-strand breaks about 40
Specific Locus test(mice) – Cell killing does not correlate with single-
strand breaks
• e.g. H2O2 produce single-strand breaks
efficiently, very few double strand breaks →
kill very few cells
– Cell killing relates better to double-strand
Genetic effect
Introduction
Structural changes
Chromosomes
and
Chromosome breakage
Breakage hypotheses
Gene mutations
Cell Division

Chromosomes and Cell Division

Genetic effect
Introduction
Structural changes
Chromosome breakage
Breakage hypotheses
Gene mutations

Illustration cells sizes of the DNA helix, the various stages of folding
of the relative
and packing of the DNA, and an entire chromosome condense at metaphase


Genetic effect • Interphase
– Largest part of the life of any somatic cell
Introduction
– Nucleus (in a stained preparation)
Structural changes
• Lacework of fine, lightly stained material in a
Chromosome breakage
translucent, colorless material surrounded by a
Breakage hypotheses membrane
• Nucleoli (1 or more bodies of various sizes &
Gene mutations shapes)
Muller’s Sex-linked test • Quantity of DNA in the nucleus doubles
Specific Locus test(mice) • Each chromosome lays down an exact replica
of itself next to itself
– Chromosome are not free to move within
the nucleus, but are restricted to “domain”
Chromosome and Cell Division

Genetic effect • Prophase
– First phase of division
Introduction
– Thickening of the chromatin
Structural changes
Chromosome breakage
– Increase stainability (chromosomes
Breakage hypotheses
condense into light coils)
– Centromere
Gene mutations • Lightly staining constriction
Muller’s Sex-linked test • End of prophase
Specific Locus test(mice) – End of the prophase
• Chromosome reach maximal condensation
Gene mutation in cultured mammalian cells membrane and nucleoli disappears
• Nuclear


Genetic effect • Metaphase
– The chromosome move to the center of the
Introduction cell (to the cell’s equator)
Structural changes – Forms spindle
Chromosome breakage • Composed of fibers that cross the cell
Breakage hypotheses • Linking its poles
– Centromeres divide
Gene mutations
• Anaphase
– Movement of the chromosomes on the
spindle to the poles
• mammalian
Gene mutation in cultured Telophasecells
– Chromosome congregated at the poles of
the cell
RADIATION– Begin to uncoil
BIOPHYSICS.1990 E. ALPEN 62
Genetic effect
Introduction
Structural changes
Chromosome breakage The Role of Telomeres
Breakage hypotheses
Gene mutations

The Role of Telomeres

Genetic effect • Telomere
– Literally means “end part”
Introduction – Cap & protect the terminal ends of
Structural changes chromosomes
Chromosome breakage – Consist long array of TTAGGG repeats
Breakage hypotheses – Range in total length from 1.5 – 150
Kilobases
Gene mutations
• In normal somatic cell divides
– Lost telomeric DNA from lagging strand in
each time
– Successive
Gene mutation in cultured mammalian cells divisions lead to progressive
shortening
– After 40 – 60 divisions, vital DNA
sequence begin to lost

Genetic effect • In normal somatic cell divides (con’t)
– Telomere length → “molecular clock” or
Introduction generation clock
Structural changes • Shortens with age in somatic tissue cells during
adult life
Chromosome breakage
– In stem cell or cancer cell
Breakage hypotheses
• Avoid the problem of aging by activating the
Gene mutations enzyme telomerase
Muller’s Sex-linked test • Telomerase
Specific Locus test(mice) – Reverse transcriptase
– Includes the complementary sequence to
the TTAGGG
Gene mutation in cultured mammalian cells repeats
– Continue rebuilds the chromosome ends
– Offset the degradation in each division

Genetic effect • In tissue culture
– Immortalization of the cells is associated
Introduction with
Structural changes • Telomere stabilization
Chromosome breakage • Activity of telomerase
Breakage hypotheses • Hypothesis: Both immortalization and
carcinogenesis are associated with
Gene mutations
telomerase expression
– Telomerase activity (+)
• All human tumor-cell lines
• 90% of human cancer biopsy
– No telomerase activity
• Normal human somatic cell tissue

Genetic effect
Introduction
Structural changes
Chromosome breakage Radiation-induced
Breakage hypotheses
Chromosome Aberrations
Gene mutations

Radiation-induced Chromosome Aberrations

Genetic effect • Difficulties in studying of radiation
damage in mammalian cell
Introduction
– Large number of chromosome per cell
Structural changes
• Diploid complement > 40 chromosome
Chromosome breakage
• Exception:
Breakage hypotheses – Chinese hamster → 22 chromosomes
– Rat kangaroo → 12 chromosomes
Gene mutations
– Woolly opossum → 14 chromosomes
– Small size
• Plant cell used recently
– Fewercells
Gene mutation in cultured mammalian and large chromosomes


Genetic effect • The change of chromosomes after x-
rays irradiated to cells
Introduction – Breaks are produced
Structural changes – The broken ends appear “sticky”
Chromosome breakage – Can rejoin with any other sticky end
Breakage hypotheses – Different fragments may behave in a
Gene mutations variety of ways
• The breaks may restitute, rejoin in their original
Muller’s Sex-linked test configuration
Specific Locus test(mice) – Nothing amiss is visible at the next mitosis
• The breaks may fail to rejoin → aberration
Gene mutation in cultured mammalian– cells
Score as a deletion at the next mitosis
• Broken ends may reassort and rejoin other
broken ends
– Chromosomes appear to be grossly distorted

Genetic effect • Types of aberration
Introduction – Chromosome aberration
Structural changes • Irradiated early in interphase
Chromosome breakage • Before chromosome material has been
Breakage hypotheses duplicated
• Break is in a single strand chromatin
Gene mutations
• In DNA synthetic phase → replicated
the break
• Identical break in the corresponding
points
Gene mutation in cultured mammalian cellsof a pair of chromatin strands


Genetic effect • Types of aberration
Introduction – Chromatid aberrations
Structural changes • Irradiated later in interphase
Chromosome breakage • After DNA material has doubled
Breakage hypotheses • The chromosomes consisted of 2
strands of chromatin
Gene mutations
• Breaks one chromatid without breaking
its sister chromatid (at least not in the
same place)

Genetic effect
Introduction Examples of
Radiation-induced
Structural changes
Chromosome breakage
Breakage hypotheses
Aberrations
Gene mutations

Examples of
Radiation-induced Aberrations
Genetic effect • Three types of aberration that are
Introduction lethal to the cell
Structural changes – Chromosome aberrations
Chromosome breakage • Dicentric
Breakage hypotheses • Ring
Gene mutations – Chromatid aberration
Muller’s Sex-linked test • Anaphase bridge

Examples of
Genetic effect Dicentric
– Involved an interchange
Introduction between 2 separate
chromosomes
Structural changes – Breaks is produce in each
Chromosome breakage one early in interphase
– Sticky ends are close to one
Breakage hypotheses
another → rejoin
– Bizarre interchange is
Gene mutations
replicated in DNA synthetic
Muller’s Sex-linked test phase
Specific Locus test(mice) – Result:
• grossly distorted
chromosome with 2
centromerecells
(dicentric)
• A fragment that has no
centromere (acentric)

Examples of
Genetic effect
Introduction
Structural changes
Chromosome breakage
Breakage hypotheses
Gene mutations
Normal metaphase Dicentric and fragment
Radiation-induced chromosome aberrations in human leukocytes
viewed at metaphase.
Examples of
Genetic effect • Ring
– Break in each arm
Introduction of a single
Structural changes chromatin early in
cell cycle
Chromosome breakage
– Sticky ends may
Breakage hypotheses rejoin to form a ring
and a fragment
Gene mutations – Acentric fragment
Muller’s Sex-linked test will be lost at
Specific Locus test(mice) mitosis (will not
pulled to either pole
of the cell

Examples of
Genetic effect
Introduction
Structural changes
Chromosome breakage
Breakage hypotheses
Gene mutations

Ring
Radiation-induced chromosome aberrations in human leukocytes
viewed at metaphase.
Examples of
Genetic effect • Anaphase bridge
– Break occur late in
Introduction cell cycle (in G2)
– After chromosome
Structural changes
have replicated
Chromosome breakage – Breaks occur in both
Breakage hypotheses chromatids of the
same chromosome
Gene mutations – Sticky ends rejoin to
form a sister union
– At anaphase, two
Specific Locus test(mice) centromeres is
stretched across the
cell between
Gene mutation in cultured mammalian cells 2 poles
– Hindering the
separation into 2
new daughter cells
Examples of
Genetic effect
Introduction
Structural changes
Chromosome breakage
Breakage hypotheses
Gene mutations
Specific Locus test(mice) Normal anaphase Bridge and fragment
resulting from
Gene mutation in cultured mammalian cells radiation
Anaphase chromosome preparation of Tradescantia paludosa

Examples of
Genetic effect • Two types of chromosomal
Introduction changes that are not lethal to the
Structural changes cell
Chromosome breakage – Symmetric translocation
Breakage hypotheses
– Small deletions
Gene mutations

Examples of
Genetic effect • Symmetrical
translocation
Introduction – Involve a break in 2
prereplication
Structural changes chromosomes (G1-
Chromosome breakage phase)
Breakage hypotheses
– Broken ends being
exchanged between
the 2 chromosomes
Gene mutations Illustration of the formation of a
– Easy to observe with symmetrical translocation
Muller’s Sex-linked test the technique of
Specific Locus test(mice) fluorecent in situ
hybridization
(chromosome
Gene mutation in cultured mammalian painting) cells
– May lead to activiation
of an oncogene → e.g.
Burkitt’s lymphoma
Examples of
Genetic effect Small interstitial
deletion
Introduction – Involved 2
breaks in the
Structural changes same arm of the
Chromosome breakage same
chromosome
Breakage hypotheses
– Loss of the
genetic
Gene mutations information
Muller’s Sex-linked test between the 2
Specific Locus test(mice) breaks
– May associated
with
carcinogenesis
• Lost
Illustration of the formation of a deletion by
suppressor ionizing radiation in an interphase chromosome.
gene
Examples of
Genetic effect • The interaction between breaks in
Introduction
different chromosomes
Structural changes
– Sites and chromosome dependence
Chromosome breakage
• e.g. Chromosome 8 → particular
sensitive to exchanges
Breakage hypotheses
– Most interaction occur at the edges
Gene mutations of domains
Muller’s Sex-linked test • Each chromosome is restricted to a
Specific Locus test(mice) domain
• Involves the nuclear matrix
Gene mutation in cultured mammalian cells chromosomes → biggest surface
• Active
area to their domains
Genetic effect
Introduction
Structural changes
Chromosome Aberrations
Chromosome breakage
in
Breakage hypotheses Human Lymphocytes
Gene mutations

Chromosome Aberrations in
Human Lymphocytes
Genetic effect • Biomarkers of radiation exposure
– Blood samples obtained after total-body
Introduction
irradiation (within a few days to a few
Structural changes
weeks)
Chromosome breakage • Frequency of asymmetric aberration in
Breakage hypotheses lymphocyte = dose received
– Stimulated to divide with a mitogen (e.g.
Gene mutations
phytohemagglutinin)
– Arrest at metaphase
– Incident of rings & dicentrics is scored
– Comparison
Gene mutation in cultured mammalian cells with in vitro cultures exposed
to known dose
Chromosome Aberrations in Human

Lymphocytes
Genetic effect Data are fitted by a
linear-quadratic
Introduction relationship
Structural changes – Rings & dicentric
Chromosome breakage
result from the
interaction of 2
Breakage hypotheses chromosome breaks
Gene mutations – Linear component
• 2 breaks resulting
from a charged particle
– Quadratic component
• 2 breaks resulting from
different charged
particles

Human Lymphocytes
(Con’t)
Introduction – Cytogenetic evaluations in cultured
Structural changes lymphocytes
Chromosome breakage • Can detect a recent total-body exposure of 0.25
Gy in the exposed person
Breakage hypotheses
• Useful in distinguishing “real” and “suspected”
Gene mutations exposure of high risk person (e.g. Black film
badge)
Muller’s Sex-linked test • Limitation
Specific Locus test(mice) – The yield of dicentrics declines in months and
years after a radiation exposure
– A finite lifespan: 1500 days
Gene mutation in cultured mammalian– cells
Dicentric are referred to as “unstable”
aberrations

Human Lymphocytes
(Con’t)
Introduction – Symmetric translocation
Structural changes • referred as “stable” aberration
Chromosome breakage • Survive and pass on the aberration to their
progeny
Breakage hypotheses
• Give accurate pictures of irradiation for many
Gene mutations years
• Fluorescent in situ hybridization technique
make the scoring more simple
Specific Locus test(mice) • Frequency of translocation correlates with total-
body ;dose in exposed persons even after more
than 50 years
– e.g. study of the survivors of atomic-bomb
attacks on Hiroshima & Nagasaki

Active listener support lecturer

Genetic effect
Introduction
Structural changes
Chromosome breakage
Breakage hypotheses
Gene mutations

The correct decision could be found only if all
members of the group actively participate in a
discussion, and use knowledge they have
received during a lecture.
Genetic effect
Introduction
Structural changes
Chromosome breakage
Breakage hypotheses
Gene mutations

Diego Rivera

Happy
chromosomal
couple!!!
Genetic effect
Introduction
Structural changes
Chromosome breakage
Breakage hypotheses
Gene mutations

Genetics

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Stochastic Effects:Genetic Effects of Ionizing Radiation

RADIATION BIOPHYSICS.1990 E. ALPEN 2

Gene mutation in cultured mammalian cells

RADIATION BIOPHYSICS.1990 E. ALPEN 5

Gene mutations  Breakage before DNA replication

RADIATION BIOPHYSICS.1990 E. ALPEN 8

RADIATION BIOPHYSICS.1990 E. ALPEN 10

Single hit breakage

RADIATION BIOPHYSICS.1990 E. ALPEN 11

Gene mutation in cultured mammalian cells

RADIATION BIOPHYSICS.1990 E. ALPEN 12

Gene mutation in cultured mammalian cells

RADIATION BIOPHYSICS.1990 E. ALPEN 14

Gene mutation in cultured mammalian cells

RADIATION BIOPHYSICS.1990 E. ALPEN 15

RADIATION BIOPHYSICS.1990 E. ALPEN 16

Gene mutation in cultured mammalian cells

RADIATION BIOPHYSICS.1990 E. ALPEN 21

RADIATION BIOPHYSICS.1990 E. ALPEN 22

Muller’s sex-linked recessive test

Muller’s sex-linked recessive test

RADIATION BIOPHYSICS.1990 E. ALPEN 24

Muller’s sex-linked recessive test

RADIATION BIOPHYSICS.1990 E. ALPEN 25

Muller’s sex-linked recessive test

RADIATION BIOPHYSICS.1990 E. ALPEN 26

Muller’s sex-linked recessive test

Chromosome breakage Y Y/C l B Y/+

RADIATION BIOPHYSICS.1990 E. ALPEN 27

Muller’s sex-linked recessive test

RADIATION BIOPHYSICS.1990 E. ALPEN 28

Muller’s sex-linked recessive test

RADIATION BIOPHYSICS.1990 E. ALPEN 29

Conclusion Muller’s sex-linked recessive test

RADIATION BIOPHYSICS.1990 E. ALPEN 30

Conclusion Muller’s sex-linked recessive test

Gene mutation in cultured mammalian cells

RADIATION BIOPHYSICS.1990 E. ALPEN 31

Conclusion Muller’s sex-linked recessive test

Specific locus test in mice

RADIATION BIOPHYSICS.1990 E. ALPEN 33

Specific locus test in mice

RADIATION BIOPHYSICS.1990 E. ALPEN 34

Gene mutation in cultured mammalian cells

Specific locus test in mice

Gene mutation in cultured mammalian cells

Specific locus test in mice

RADIATION BIOPHYSICS.1990 E. ALPEN 37

Specific locus test in mice(male)

Gene mutation in cultured mammalian cells

RADIATION BIOPHYSICS.1990 E. ALPEN 38

Specific locus test in mice(female)

Gene mutation in cultured mammalian cells

RADIATION BIOPHYSICS.1990 E. ALPEN 39

Mutation in Cultured mammalian cell

DNA Strand Breaks

Gene mutation in cultured mammalian cells

RADIATION BIOPHYSICS.1990 E. ALPEN 41

DNA Strand Break

RADIATION BIOPHYSICS.1990 E. ALPEN 42

DNA Strand Breaks

RADIATION BIOPHYSICS.1990 E. ALPEN 43