Genetics

Stochastic Effects:Genetic Effects of Ionizing Radiation

Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

Stochastic Effects

genetic effects of ionizing radiation 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

Stochastic Effects:Genetic Effects of Ionizing Radiation

Introduction Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

Gene mutations Muller’s Sex-linked test Specific Locus test(mice)

• The change in DNA of the gametes (Genetic Mutation)are indeed of the stochastic class. • They occur on probabilistic basis with no threshold for the occurrence and the severity of the phenotypic changes resulting from them is unrelated to dose.

Gene mutation in cultured mammalian cells

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Introduction Genetic effect

• Loss of clonogenic potential is due to defect in the DNA of the cell Introduction 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 Muller’s Sex-linked test gametes ,it may be passed on to Specific Locus test(mice) future generations at the time of Gene mutation in cultured mammalian cells reproductive activity. RADIATION BIOPHYSICS.1990

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Introduction Genetic effect

• Genetic mutation Introduction →Somatic mutation Structural changes →Not transmissible to offspring Chromosome breakage →Altered function or phenotype Breakage hypotheses →An important class is CANCER Gene mutations Muller’s Sex-linked test • At that time only we could study Specific Locus test(mice) metaphase chromosomes.then the Gene mutation in cultured mammalian changecells classified in two class according to visibility . RADIATION BIOPHYSICS.1990

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Introduction Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

Gene mutations

• Either gamete or somatic dividing cell,to express the changes that have occurred in its genome must be capable of division.

Muller’s Sex-linked test Specific Locus test(mice)

Gene mutation in cultured mammalian cells

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Structural changes in chromosome • 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 production of larva that is examined. Gene mutations • Bender and Gooch(1962) Muller’s Sex-linked test 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. Genetic effect

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Structural changes in chromosome Genetic effect

• Metaphase chromosome represents only a brief portion of the life Introduction cycle of the chromosome. Structural changes Chromosome breakage • During this “invisible”period Breakage hypotheses much of importance is happening. Gene mutations • Waldren and Johnson(1974) Muller’s Sex-linked test Specific Locus test(mice) • New techniques developed for the examination of interphase Gene mutation in cultured mammalian cells chromosome. RADIATION BIOPHYSICS.1990

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Chromosome breakage Genetic effect

Structural changes

• Ionizing radiations is the most effective cause in producing this class of structural change.

Chromosome breakage

•

Introduction

Breakage hypotheses

A little consequence as a result of radiation damage is 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 (or it will failed the attempted replication)

Specific Locus test(mice)

 Breakage after DNA replication

Gene mutation in cultured mammalian cells Asymmetric change in one of the two chromatids. RADIATION BIOPHYSICS.1990

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Chromosome breakage Genetic effect

•

Time from the irradiation event to the collection of mitosis will Introduction determine the position of the cell Structural changes Chromosome breakage cycle that is being observed. Breakage hypotheses • Three types: Gene mutations • Subchromatid (prophase-hard to Muller’s Sex-linked test distinguish) Specific Locus test(mice) • Chromatid (post replication G2) Gene mutation in cultured mammalian cells • Chromosome (pre synthesis G1) RADIATION BIOPHYSICS.1990

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Chromosome breakage Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

Gene mutations Muller’s Sex-linked test Specific Locus test(mice)

• Waldern and Johnson(1974) • The majority of the breaks will be restored to their normal condition(restitution) • Rejoining to a site that is incorrect causing a transposition of nucleotide • Dalrymple(1973) Alphabetic marker indicates position on unreplicated chromatide.

Gene mutation in cultured mammalian cells

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Single hit breakage Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

A B C.D E F G H I

Gene mutations Muller’s Sex-linked test Specific Locus test(mice)

A B C .D E F and G H I

Gene mutation in cultured mammalian cells

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Inversion of the fragment or rejoining at the wrong end Genetic effect

Introduction Structural changes Chromosome breakage

• A B C.D E F I H G • I H G A B C .D E F • G H I A B C .D E F

Breakage hypotheses

Gene mutations Muller’s Sex-linked test Specific Locus test(mice)

Gene mutation in cultured mammalian cells

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Two-hit breakage Genetic effect

Introduction

A B C.D E F G H I

Structural changes Chromosome breakage

Breakage hypotheses

Gene mutations Muller’s Sex-linked test Specific Locus test(mice)

A B C.D, E F and G H I A B C.D G H I Interstitial deletion and EF is a minute

E F A B C .D I H G

Gene mutation in cultured mammalian cells

Inversion reordering without loss RADIATION BIOPHYSICS.1990

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Multiple hits in replicated chromosome Genetic effect

Introduction

• Exchange of parts of the DNA between the two chromatids or chromosome is a possibility .

Structural changes Chromosome breakage

Breakage hypotheses

Isocentric break

Gene mutations Muller’s Sex-linked test Specific Locus test(mice)

Gene mutation in cultured mammalian cells

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Multiple hits in replicated chromosome Genetic effect

Introduction

• • •

Structural changes

•

Chromosome breakage

Dicentric product Movement of parts is called “translocation” If all genetic info. Is intact it called”balanced translocation”. Chemical damage leads to “sister chromatid exchange”

Dicentric chromosome

Breakage hypotheses

Gene mutations Muller’s Sex-linked test Specific Locus test(mice)

Gene mutation in cultured mammalian cells

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Breakage hypothesis Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

Gene mutations Muller’s Sex-linked test Specific Locus test(mice)

• Sax (1940) • Lea’s target theory:  Aberrations that requires one break(hit)should follow the dose response relationship that is first order in dose.  Aberrations that requires two or three breaks(hits)should follow the dose response relationship that is respectively second or third order in dose.

Target model has not entirely explain the Gene mutation in cultured mammalian cells data for chromosome aberration. RADIATION BIOPHYSICS.1990

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Breakage hypothesis • Revell(1955) • “achromatic gap”non-staining Introduction region of the chromatid the D-R Structural changes curve was first order in dose. Chromosome breakage Breakage hypotheses • On the other hand the frequency of true chromatid deletions,which Gene mutations 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. Gene mutation in cultured mammalian cells • “Exchange hypothesis” Genetic effect

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Exchange hypothesis • All chromosome aberrations arise from an exchange process that has Introduction much in common with,and many Structural changes be similar to,meiotic crossing over Chromosome breakage that occurs normally during the Breakage hypotheses division stage of the gamete. Gene mutations • The production of chromosome Muller’s Sex-linked test aberration is very complicated. Specific Locus test(mice) • The simple breakage model must Gene mutation in cultured mammalian cells ,to great extent, be put aside. Genetic effect

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Gene mutation • 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. • Many more subtle changes occur that Gene mutations are not visible in the morphology but Muller’s Sex-linked test their presence known through alteration Specific Locus test(mice) of some genetically controlled function of the cell.these are Gene mutation in cultured mammalian cells Genetic effect

”gene mutation” RADIATION BIOPHYSICS.1990

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Gene mutation

Muller’s Sex-linked test

• The small changes are called”point mutations” they give raised to a change in a codon that will miss read for a single amino acid. • Second important type of mutation can arise as the result of minor deletion,addition, or substitution called”frame shift”.

Specific Locus test(mice)

Using nonsense codon CAT.

Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

Gene mutations

• CAT CAT CAT CAT CAT CAT... Gene mutation in cultured mammalian cells

CA Deleted

• CAT CAT CAT CAT TCA TCA... RADIATION BIOPHYSICS.1990

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Gene mutation Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

Gene mutations Muller’s Sex-linked test Specific Locus test(mice)

• In the absence of radiation ,the mutation rate for a single gene may be on the order of one mutation or less per gene site per million cells that are formed • Radiation can easily raise this rate by a thousand fold or more.

Gene mutation in cultured mammalian cells

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Gene mutation Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

Gene mutations

• DeSerres,Milligan, and Wbber(1967) • They studied two adjacent gene producing Adenine.

Yield=0.3+5.39D+0.16 D² (mutation per million surviving cells),D in kilorad=10Gy

• Point mutation required one hit and Specific Locus test(mice) chromosome deletion required two hits. • This formula provide interesting insight Gene mutation in cultured mammalian cells to L-Q molecular model Muller’s Sex-linked test

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Stochastic Effects:Genetic Effects of Ionizing Radiation

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. Muller’s Sex-linked test • For being the first to show that Specific Locus test(mice) radiation could produce mutation Gene mutation in cultured mammalian cells events. RADIATION BIOPHYSICS.1990

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Muller’s sex-linked recessive test Genetic effect

• Muller(1927)

Introduction Structural changes Chromosome breakage

Breakage hypotheses

Gene mutations Muller’s Sex-linked test

Nobel Prize

Specific Locus test(mice)

Gene mutation in cultured mammalian cells

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Muller’s sex-linked recessive test Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

Gene mutations

• • • •

X/X female fly C only procedural interest l lethal gene B morphological marker

C Suppresses the crossing over B Bar-eyed to identify flies that carry the l gene

Muller’s Sex-linked test

 C l B/+ (+ is sufficient, if present,to prevent the lethal but recessive action of l gene) Gene mutation in cultured mammalian cells

Specific Locus test(mice)

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Muller’s sex-linked recessive test Genetic effect

•

Introduction Structural changes Chromosome breakage

•

Breakage hypotheses

Gene mutations Muller’s Sex-linked test Specific Locus test(mice)

•

Cross the C l B/+ female with wild type males that have been irradiated at dose chosen by the investigator. The competent, wild-type male is coded +/Y to indicate that it also has an X chromosome (+), which will prevent the action of l. Not suffered a radiation induced mutation in the X chromosome:

Gene mutation in cultured cells 4) mammalian bar-eyed daughters C l B/+ 5)

+/+ female will not bar-eyed and excluded

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Muller’s sex-linked recessive test Genetic effect

Introduction

C l B/+ +/Y

ClB

+(lm)

C l B/+(lm)  Bar-eyed

+/+ 

Y/C l B dead

Y/+

C l B/+(lm) +/Y

ClB

+(lm)

+

C l B/+  Bar-eyed

Structural changes Chromosome breakage

Y

Breakage hypotheses

Gene mutations Muller’s Sex-linked test Specific Locus test(mice)

Gene mutation in cultured mammalian cells Y

RADIATION BIOPHYSICS.1990

Y/C l B dead

+

not bar-eyed

not bar-eyed

+/+  not bar-eyed

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Muller’s sex-linked recessive test Genetic effect

• No male survive from this cross (table 2). Introduction 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 Muller’s Sex-linked test of such crosses lacking male Specific Locus test(mice) survivors is the number of original Gene mutation in cultured mammalian cells males with mutations. irradiated RADIATION BIOPHYSICS.1990

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Muller’s sex-linked recessive test Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

Gene mutations Muller’s Sex-linked test Specific Locus test(mice)

• this elegant experience allowed Muller to construct dose-effect curves for this mutation in D.M. • Muller recessive lethal model doesn’t permit us to examine the sensitivity of female for radiations mutogenesis. • The conclusion is as Follow

Gene mutation in cultured mammalian cells

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Conclusion Muller’s sex-linked recessive test Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

Gene mutations Muller’s Sex-linked test Specific Locus test(mice)

1. From 25-4000 rad the rate of production of sex-linked recessive lethal is constant per unit dose. 2. There appears to be no threshold below which an effect would not been predicted. 3. There is no effect of dose rate over a wide range .there is also no effect of fractionation of the dose .

Gene mutation in cultured mammalian cells

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Conclusion Muller’s sex-linked recessive test Genetic effect

Introduction Structural changes Chromosome breakage

1. The sensitivity of the male gamete varies greatly with the stage of development at which it is irradiated

Breakage hypotheses

Gene mutations Muller’s Sex-linked test Specific Locus test(mice)

Gene mutation in cultured mammalian cells

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Conclusion Muller’s sex-linked recessive test Genetic effect

5.The relative sensitivity of the female gamete has been measured Introduction by another method (specific Structural changes Chromosome breakage locus)the value are as follow: Breakage hypotheses oocytes and oogonia 1 Gene mutations late oocytes 2 Muller’s Sex-linked test Specific Locus test(mice) 6.The induction of mutation by inoizing radiation in DM will Gene mutation in cultured mammalian cells occur at the rate of 1.5-8 X 10^-8 RADIATION BIOPHYSICS.1990

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Specific locus test in mice Genetic effect

• Concern about the reliability of Drosophila data for projections to Introduction human beings led to the Structural changes Chromosome breakage establishment in the early 1960s of Breakage hypotheses a large project as Oak Ridge Gene mutations National Laboratory (ORNL) to Muller’s Sex-linked test establish the sensitivity of a Specific Locus test(mice) mammalian species for radiation mutagenesis . Gene mutation in cultured mammalian cells RADIATION BIOPHYSICS.1990

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Specific locus test in mice • 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 for this purpose. Gene mutations 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 Gene mutation in cultured mammalian cells or ”kinky tail” in mouse. Genetic effect

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

Gene mutations Muller’s Sex-linked test Specific Locus test(mice)

Gene mutation in cultured mammalian cells

Hope is eternal

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Specific locus test in mice Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

Gene mutations Muller’s Sex-linked test Specific Locus test(mice)

Gene mutation in cultured mammalian cells The longer we wait before the mating,the earlier will be the stage of maturation of the gamete RADIATION BIOPHYSICS.1990

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Specific locus test in mice Genetic effect

 For the seven of loci studied in the mouse the average mutation rate per Introduction locus ,per rad(per cGy) was found to Structural changes Chromosome breakage be 2.2 X 10^-7 Breakage hypotheses  Study with male mice were Gene mutations undertaken at dose rate from 0.009Muller’s Sex-linked test 90 rad/min showed repair process Specific Locus test(mice) dose-rate,not dose,dependent and Gene mutation in cultured saturated mammalian cells at 0.009 rad/min dose rate. RADIATION BIOPHYSICS.1990

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Specific locus test in mice(male) Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

Gene mutations Muller’s Sex-linked test Specific Locus test(mice)

Gene mutation in cultured mammalian cells

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Specific locus test in mice(female) Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

Gene mutations Muller’s Sex-linked test Specific Locus test(mice)

Gene mutation in cultured mammalian cells

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Mutation in Cultured mammalian cell Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

Gene mutations Muller’s Sex-linked test Specific Locus test(mice)

Deserres, Malling, and Webber(1967) • Mutation rates in fungal organism. • In the last two decades similar studies conduct for mammalian cells .(not for mammalian gamete but for somatic cells)in three types: 4) 5) 6)

Auxotrophic mutants Temperature sensitive mutants Certain drug resistant

Mutation frequencies for cultured somaticcells mammalian cells are essentially Gene mutation in cultured mammalian identical to mouse specific locus mutation frequency (1.5-3 X 10^-7) 

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

DNA Strand Breaks

Gene mutations Muller’s Sex-linked test Specific Locus test(mice)

Gene mutation in cultured mammalian cells

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Stochastic Effects:Genetic Effects of Ionizing Radiation

DNA Strand Break Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

Gene mutations Muller’s Sex-linked test Specific Locus test(mice)

• DNA is the principal target for the biological effects of radiation – Cell killing – Mutation – Carcinogenesis

• Result breaks in DNA, caused by:

– Charged-particle tracks Gene mutation in cultured mammalian cells species produced – Chemical RADIATION BIOPHYSICS.1990

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Stochastic Effects:Genetic Effects of Ionizing Radiation

DNA Strand Breaks Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

Gene mutations Muller’s Sex-linked test

• DNA – Deoxyribonucleic acid – Double helix structure • 2 strands → held together by hydrogen bone between the bases • “Backbone” of each strand → alternating sugar + phosphate group • 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) Gene mutation in cultured mammalian cells – Double ring group → Purines (e.g. adenine & guanine) RADIATION BIOPHYSICS.1990

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Stochastic Effects:Genetic Effects of Ionizing Radiation

structure The of a single strand of DNA Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

Gene mutations Muller’s Sex-linked test Specific Locus test(mice)

Gene mutation in cultured mammalian cells

The structure of a single strand of DNA RADIATION BIOPHYSICS.1990

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Stochastic Effects:Genetic Effects of Ionizing Radiation

DNA Strand Break Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

• DNA (Con’t) – Base on the opposite strands must be complementary – Adenine (A)  Thymine (T) – Guanine (G)  Cytosine (C)

Gene mutations Muller’s Sex-linked test Specific Locus test(mice)

Gene mutation in cultured mammalian cells

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Stochastic Effects:Genetic Effects of Ionizing Radiation

DNA Strand Breaks Genetic effect

Introduction Structural changes Chromosome breakage

• The cell are irradiated with x-rays – Many breaks of a single strand • Little biological consequence • Repair readily using the opposite strand as a template • Mutation occurred if the repair is incorrect

Breakage hypotheses

Gene mutations Muller’s Sex-linked test Specific Locus test(mice)

–Both strands are broken, breaks are well separated •Also repair readily •Two breaks are handled separately

Gene mutation in cultured mammalian cells

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Stochastic Effects:Genetic Effects of Ionizing Radiation

DNA Strand Breaks Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

Gene mutations Muller’s Sex-linked test

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

Specific Locus test(mice)

Gene mutation in cultured mammalian cells

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Stochastic Effects:Genetic Effects of Ionizing Radiation

DNA Strand Breaks Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

Gene mutations Muller’s Sex-linked test Specific Locus test(mice)

• Double-strand breaks – Types: • Distance between the breaks on the two DNA strands • Kinds of end groups formed

– Yield: 0.04 x of single-strand breaks – Induced linearly with dose • Formed by single tracks of ionizing radiation

Gene mutation in cultured mammalian cells

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Stochastic Effects:Genetic Effects of Ionizing Radiation

DNA Strand Breaks Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

Gene mutations Muller’s Sex-linked test Specific Locus test(mice)

– Repair • Homologous recombination – Requiring an undamaged DNA strand as participant in the repair – End-to end rejoining via nonhomologous recombination – Error free process – Rare in mammalian cells – carried out by proteins similar to rad51 gene (product of yeast S.cerevisiae)

Gene mutation in cultured mammalian cells

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Stochastic Effects:Genetic Effects of Ionizing Radiation

DNA Strand Breaks Genetic effect

Introduction

– Repair • Nonhomologous recombination

– Illegitimate recombination – Account for many of the Structural changes premutagenic lesions induced Chromosome breakage in the DNA of human cells by ionizing radiation Breakage hypotheses – Participated protein Gene mutations » DNA-dependent kinase » Ku protein Muller’s Sex-linked test – Protein complex (recent Specific Locus test(mice) research) » hMre11, Hrad50 Gene mutation in cultured mammalian cells(the product of the » p95 NBS1 gene) RADIATION BIOPHYSICS.1990

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Stochastic Effects:Genetic Effects of Ionizing Radiation

DNA Strand Breaks Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

Gene mutations Muller’s Sex-linked test Specific Locus test(mice)

• Double-strand breaks (Con’t) – Ionizing radiation • Energy is not deposited uniformly in the absorbing medium • Located along the tracks of the charged particles – Electrons → x-rays or γ-rays – α-particles → neutrons

• “Spur”, “Blobs” and “short tracks”

Gene mutation in cultured mammalian cells

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Stochastic Effects:Genetic Effects of Ionizing Radiation

DNA Strand Breaks Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

Gene mutations Muller’s Sex-linked test Specific Locus test(mice)

•

Ionizing radiation (Con’t) – “Spur” • • • •

Contain up to 100 eV Involved 3 ion pairs 95% of energy deposition by x- or γ-rays Diameter: 4 nm (2x diameter of the DNA double helix)

– “Blobs” • Less frequent for x- or γ-rays • Frequent in densely ionizing radiation (e.g. neutrons, αparticle) • Diameter: 7 nm • Involved 12 ion pairs • More difficult for the cell repair

– “Spurs” & “blobs” have dimension similar to DNA double helix

• → multiple Gene mutation in cultured mammalian cells radical attack occurs if they overlap the DNA helix. • Wide variety of complex lesions • Base damage RADIATION BIOPHYSICS.1990 • Double strand breaks

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

Gene mutations Muller’s Sex-linked test Specific Locus test(mice)

Illustration of a locally multiply damage site

• Locallycells multiply Gene mutation in cultured mammalian

damaged site

– Multiple damage spread out up to 20 base pairs RADIATION BIOPHYSICS.1990

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

Measuring DNA Strand Breaks

Gene mutations Muller’s Sex-linked test Specific Locus test(mice)

Gene mutation in cultured mammalian cells

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Measuring DNA Strand Breaks Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

Gene mutations Muller’s Sex-linked test

• Isolating the DNA from irradiated cell – DNA pieces to pass through a porous substrate (e.g. gel, filter) – DNA pieces move under the influence of • • • •

Gel → electric field Filter → flow Small pieces → faster & farther Large pieces → slower & nearer

– Measuring single strand breaks → strong alkaline preparation Gene mutation in cultured mammalian cells double strand breaks → neutral – Measuring preparation

Specific Locus test(mice)

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Measuring DNA Strand Breaks Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

Gene mutations Muller’s Sex-linked test Specific Locus test(mice)

• DNA in cell is more resistant to damage by radiation (c.f. free DNA) – Presence of low molecular weight scavenger in cells • Mop up some of the free radicals

– Physical protection by packaging • Sequence-specific sensitivity • Actively translating genes → more sensitive to radiation

Gene mutation in cultured mammalian cells

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Measuring DNA Strand Breaks Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

Gene mutations Muller’s Sex-linked test Specific Locus test(mice)

• D0 dose – One lethal event / cell – 37 % still viable – e.g. mammalian cell → D0 lies between 1 – 2 Gy • Number of DNA lesions per cell – Base damage > 1000 – Single-strand breaks about 1000 – Double-strand breaks about 40

– Cell killing does not correlate with singlestrand breaks

• e.g. H2O2 produce single-strand breaks Gene mutation in cultured mammalian cells efficiently, very few double strand breaks → kill very few cells

– Cell killing relates better to double-strand

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

Gene mutations

Chromosomes and Cell Division

Muller’s Sex-linked test Specific Locus test(mice)

Gene mutation in cultured mammalian cells

Stochastic Effects:Genetic Effects of Ionizing Radiation

Chromosomes and Cell Division Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

Gene mutations Muller’s Sex-linked test Specific Locus test(mice)

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

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Chromosomes and Cell Division Genetic effect

Introduction Structural changes

• Interphase – Largest part of the life of any somatic cell – Nucleus (in a stained preparation)

• Lacework of fine, lightly stained material in a 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 Gene mutation in cultured mammalian cells – Chromosome are not free to move within Chromosome breakage

the nucleus, but are restricted to “domain” RADIATION BIOPHYSICS.1990

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Chromosome and Cell Division Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

Gene mutations Muller’s Sex-linked test Specific Locus test(mice)

• Prophase – First phase of division – Thickening of the chromatin – Increase stainability (chromosomes condense into light coils) – Centromere • Lightly staining constriction • End of prophase

– End of the prophase

• Chromosome reach maximal condensation Gene mutation in cultured mammalian cells membrane and nucleoli disappears • Nuclear

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Chromosomes and Cell Division Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

Gene mutations Muller’s Sex-linked test Specific Locus test(mice)

• Metaphase – The chromosome move to the center of the cell (to the cell’s equator) – Forms spindle • Composed of fibers that cross the cell • Linking its poles

– Centromeres divide

• Anaphase – Movement of the chromosomes on the spindle to the poles

• mammalian Telophase Gene mutation in cultured cells

– Chromosome congregated at the poles of the cell RADIATION– BIOPHYSICS.1990 E. ALPEN Begin to uncoil

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Genetic effect

Introduction Structural changes Chromosome breakage

The Role of Telomeres

Breakage hypotheses

Gene mutations Muller’s Sex-linked test Specific Locus test(mice)

Gene mutation in cultured mammalian cells

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Stochastic Effects:Genetic Effects of Ionizing Radiation

The Role of Telomeres Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

Gene mutations Muller’s Sex-linked test

• Telomere – Literally means “end part” – Cap & protect the terminal ends of chromosomes – Consist long array of TTAGGG repeats – Range in total length from 1.5 – 150 Kilobases

• 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 RADIATION BIOPHYSICS.1990 E. ALPEN

Specific Locus test(mice)

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Stochastic Effects:Genetic Effects of Ionizing Radiation

The Role of Telomeres Genetic effect

• In normal somatic cell divides (con’t)

Introduction

– Telomere length → “molecular clock” or generation clock

Structural changes Chromosome breakage

Breakage hypotheses

Gene mutations Muller’s Sex-linked test

• Shortens with age in somatic tissue cells during adult life

– In stem cell or cancer cell • Avoid the problem of aging by activating the enzyme telomerase

• Telomerase

– Reverse transcriptase – Includes the complementary sequence to the TTAGGG repeats Gene mutation in cultured mammalian cells – Continue rebuilds the chromosome ends – Offset the degradation in each division

Specific Locus test(mice)

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Stochastic Effects:Genetic Effects of Ionizing Radiation

The Role of Telomeres Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

Gene mutations Muller’s Sex-linked test Specific Locus test(mice)

• In tissue culture – Immortalization of the cells is associated with • Telomere stabilization • Activity of telomerase

• Hypothesis: Both immortalization and carcinogenesis are associated with telomerase expression – Telomerase activity (+)

• All human tumor-cell lines • 90% of human cancer biopsy Gene mutation in cultured mammalian cells –

No telomerase activity

• Normal human somatic cell tissue RADIATION BIOPHYSICS.1990

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

Radiation-induced Chromosome Aberrations

Gene mutations Muller’s Sex-linked test Specific Locus test(mice)

Gene mutation in cultured mammalian cells

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Radiation-induced Chromosome Aberrations Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

Gene mutations Muller’s Sex-linked test Specific Locus test(mice)

• Difficulties in studying of radiation damage in mammalian cell – Large number of chromosome per cell • Diploid complement > 40 chromosome • Exception: – Chinese hamster → 22 chromosomes – Rat kangaroo → 12 chromosomes – Woolly opossum → 14 chromosomes

– Small size

• Plant cell used recently

Gene mutation in cultured mammalian – Fewercells and

RADIATION BIOPHYSICS.1990

large chromosomes

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Radiation-induced Chromosome Aberrations Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

Gene mutations

• The change of chromosomes after xrays irradiated to cells – – – –

Breaks are produced The broken ends appear “sticky” Can rejoin with any other sticky end Different fragments may behave in a variety of ways

Muller’s Sex-linked test

• The breaks may restitute, rejoin in their original configuration

Specific Locus test(mice)

– Nothing amiss is visible at the next mitosis

• The breaks may fail to rejoin → aberration Score as a deletion at the next mitosis Gene mutation in cultured mammalian– cells • Broken ends may reassort and rejoin other broken ends – Chromosomes appear to be grossly distorted RADIATION BIOPHYSICS.1990

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Radiation-induced Chromosome Aberrations Genetic effect

Introduction

• Types of aberration – Chromosome aberration

• Irradiated early in interphase • Before chromosome material has been Chromosome breakage duplicated Breakage hypotheses • Break is in a single strand chromatin Gene mutations • In DNA synthetic phase → replicated Muller’s Sex-linked test the break Specific Locus test(mice) • Identical break in the corresponding points Gene mutation in cultured mammalian cellsof a pair of chromatin strands Structural changes

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Radiation-induced Chromosome Aberrations Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

Gene mutations Muller’s Sex-linked test Specific Locus test(mice)

• Types of aberration – Chromatid aberrations • Irradiated later in interphase • After DNA material has doubled • The chromosomes consisted of 2 strands of chromatin • Breaks one chromatid without breaking its sister chromatid (at least not in the same place)

Gene mutation in cultured mammalian cells

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

Gene mutations

Examples of Radiation-induced Aberrations

Muller’s Sex-linked test Specific Locus test(mice)

Gene mutation in cultured mammalian cells

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Examples of Radiation-induced Aberrations Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

Gene mutations Muller’s Sex-linked test

• Three types of aberration that are lethal to the cell – Chromosome aberrations • Dicentric • Ring

– Chromatid aberration • Anaphase bridge

Specific Locus test(mice)

Gene mutation in cultured mammalian cells

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Examples of Radiation-induced Aberrations Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

Gene mutations Muller’s Sex-linked test Specific Locus test(mice)

Dicentric – Involved an interchange between 2 separate chromosomes – Breaks is produce in each one early in interphase – Sticky ends are close to one another → rejoin – Bizarre interchange is replicated in DNA synthetic phase – Result:

Gene mutation in cultured

• grossly distorted chromosome with 2 mammalian centromerecells (dicentric) • A fragment that has no centromere (acentric)

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Examples of Radiation-induced Aberrations Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

Gene mutations Muller’s Sex-linked test Specific Locus test(mice)

Normal metaphase

Dicentric and fragment

Gene mutation in cultured mammalian cells Radiation-induced chromosome aberrations in human leukocytes viewed at metaphase. RADIATION BIOPHYSICS.1990

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Examples of Radiation-induced Aberrations Genetic effect

• Ring

– Break in each arm Introduction of a single chromatin early in Structural changes 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 mitosis (will not Specific Locus test(mice) pulled to either pole of the cell Gene mutation in cultured mammalian cells

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Examples of Radiation-induced Aberrations Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

Gene mutations Muller’s Sex-linked test Specific Locus test(mice)

Gene mutation in cultured mammalian cells

Ring

Radiation-induced chromosome aberrations in human leukocytes viewed at metaphase. RADIATION BIOPHYSICS.1990

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Examples of Radiation-induced Aberrations Genetic effect

• Anaphase bridge

– Break occur late in cell cycle (in G2) Introduction – After chromosome Structural changes have replicated Chromosome breakage – Breaks occur in both chromatids of the Breakage hypotheses same chromosome – Sticky ends rejoin to Gene mutations form a sister union Muller’s Sex-linked test – 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 RADIATION BIOPHYSICS.1990

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Examples of Radiation-induced Aberrations Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

Gene mutations Muller’s Sex-linked test Specific Locus test(mice)

Normal anaphase

Gene mutation in cultured mammalian cells

Bridge and fragment resulting from radiation

Anaphase chromosome preparation of Tradescantia paludosa RADIATION BIOPHYSICS.1990

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Examples of Radiation-induced Aberrations Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

• Two types of chromosomal changes that are not lethal to the cell – Symmetric translocation – Small deletions

Gene mutations Muller’s Sex-linked test Specific Locus test(mice)

Gene mutation in cultured mammalian cells

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Examples of Radiation-induced Aberrations Genetic effect

• Symmetrical translocation

Introduction

– Involve a break in 2 prereplication Structural changes chromosomes (G1phase) Chromosome breakage – Broken ends being Breakage hypotheses exchanged between the 2 chromosomes Gene mutations – Easy to observe with Muller’s Sex-linked test the technique of fluorecent in situ Specific Locus test(mice) hybridization (chromosome Gene mutation in cultured mammalian cells painting) – May lead to activiation of an oncogene → e.g. Burkitt’s lymphoma RADIATION BIOPHYSICS.1990

Illustration of the formation of a symmetrical translocation

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Examples of Radiation-induced Aberrations Genetic effect

Small interstitial deletion

Introduction

– Involved 2 breaks in the Structural changes same arm of the same Chromosome breakage chromosome Breakage hypotheses – Loss of the genetic Gene mutations information Muller’s Sex-linked test between the 2 breaks Specific Locus test(mice) – May associated with Gene mutation in cultured mammalian cells carcinogenesis • Lost Illustration of the formation of a deletion by suppressor ionizing radiation in an interphase chromosome. gene

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Examples of Radiation-induced Aberrations Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

Gene mutations

• The interaction between breaks in different chromosomes – Sites and chromosome dependence • e.g. Chromosome 8 → particular sensitive to exchanges

– Most interaction occur at the edges of domains

• 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 Muller’s Sex-linked test

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

Chromosome Aberrations in Human Lymphocytes

Gene mutations Muller’s Sex-linked test Specific Locus test(mice)

Gene mutation in cultured mammalian cells

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Chromosome Aberrations in Human Lymphocytes Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

• Biomarkers of radiation exposure – Blood samples obtained after total-body irradiation (within a few days to a few weeks) • Frequency of asymmetric aberration in lymphocyte = dose received

– Stimulated to divide with a mitogen (e.g. phytohemagglutinin) Muller’s Sex-linked test – Arrest at metaphase Specific Locus test(mice) – Incident of rings & dicentrics is scored Gene mutation in cultured mammalian cells with in vitro cultures exposed – Comparison to known dose Gene mutations

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Chromosome Aberrations in Human Lymphocytes Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

Gene mutations Muller’s Sex-linked test Specific Locus test(mice)

Data are fitted by a linear-quadratic relationship – Rings & dicentric result from the interaction of 2 chromosome breaks – Linear component • 2 breaks resulting from a charged particle

– Quadratic component

• 2 breaks resulting from Gene mutation in cultured mammalian cells different charged particles RADIATION BIOPHYSICS.1990

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Chromosome Aberrations in Human Lymphocytes Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

Gene mutations Muller’s Sex-linked test

• Biomarkers of radiation exposure (Con’t) – Cytogenetic evaluations in cultured lymphocytes • Can detect a recent total-body exposure of 0.25 Gy in the exposed person • Useful in distinguishing “real” and “suspected” exposure of high risk person (e.g. Black film badge) • Limitation

– 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

Specific Locus test(mice)

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Chromosome Aberrations in Human Lymphocytes Genetic effect

Introduction Structural changes

• Biomarkers of radiation exposure (Con’t) – Symmetric translocation

• referred as “stable” aberration • Survive and pass on the aberration to their Chromosome breakage progeny Breakage hypotheses • Give accurate pictures of irradiation for many years Gene mutations • Fluorescent in situ hybridization technique Muller’s Sex-linked test make the scoring more simple Specific Locus test(mice) • Frequency of translocation correlates with totalbody ;dose in exposed persons even after more than 50 years Gene mutation in cultured mammalian cells – e.g. study of the survivors of atomic-bomb attacks on Hiroshima & Nagasaki

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Stochastic Effects:Genetic Effects of Ionizing Radiation

Active listener

support lecturer

Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

Gene mutations Muller’s Sex-linked test Specific Locus test(mice)

Gene mutation in cultured mammalian cells

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Stochastic Effects:Genetic Effects of Ionizing Radiation 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 Muller’s Sex-linked test Specific Locus test(mice)

Gene mutation in cultured mammalian cells

Diego Rivera RADIATION BIOPHYSICS.1990

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90

Happy Stochastic Effects:Genetic Effects of Ionizing Radiation chromosomal couple!!! Genetic effect

Introduction Structural changes Chromosome breakage

Breakage hypotheses

Gene mutations Muller’s Sex-linked test Specific Locus test(mice)

Gene mutation in cultured mammalian cells

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