PCR assay of the molecular alternations at cinnabar and

vestigial genes of Drosophila melanogaster

after γ-rays and neutron action

• Aleksievich Olga• Sheresh Irina• Lebogang Sepini

• Igor D. Alexandrov, Ph. D., Dr. Sci. (Biology), chief. Sci. res.

• Margarita V. Alexandrova, Ph. D., senior sci. res.

GOAL OF PROJECT

TO STUDY THE MOLECULAR GENETIC ACTION OF GAMMA-RAYS AND NEUTRONS ON THE

CINNABAR AND VESTIGIAL GENES IN DROSOPHILA

MELANOGASTER GERM CELLS

Potential genetic risksNuclear explosion

Sun radiationTechnogenic catastrophes

Why Drosophila?• Well studied example,

gene structure known• Has common principal

DNA structure with humans

• Short life cycle (~15 days)

• Permits the study of heritable gene mutation

• Low cost

Wild typeCinnabar mutant

Vestigial mutant

Chromosome map

Main steps

• Isolation of DNA• Polymerase chain reaction• Electrophoresis

Isolation of DNADNAs were isolated from imago of wild type (as

control) and cinnabar and vestigial mutants using procedure below:• Cell lysis• DNA absorption from the

nucleus surface with silica solution (NucleoS™).

• Washing DNA with solution buffer

• DNA extraction from silica solution with ExtraGene™.

PCR

-to amplify specific fragment of DNA

- method based on in vitro replication of DNA using:

• Taq polymerase• primers• dNTP• reaction buffer

-allows the detection of different kinds of mutational changes in separate fragments of the gene (e.g. insertions or deletions)

Amplificator

Gel electrophoresis

Scheme of vestigial gene

Ex 6-8

Ex 5

Ex 3

Ex 4

Ex 1

Ex 2

Vestigial mutants with génotype C3G

код мутации

вид облуче ния, доза,

Гр

ex1 (983b)

ex2 (777b)

ex3 (471b)

ex4 (381b)

ex5 (670b)

ex6-8 (768b)

№ 1 3 8 10 15 16

1 307 + + + + + +

2 308 + + + + + +

3 vg85e4 γ, 20 + + + + + +

4 vg84f X, 40 + + + - + +

5 vg76d1 γ, 40 + + + + + +

6 vg78b1 γ, 40 + + + + + +

7 vg78b3 γ, 40 + + + + + +

8 vg76d2 γ, 40 + + + + + -

9 vg78a2 γ, 40 + + + + + +

10 vg85d1 n, 15 + + + + + +

Results

• 2 control lines and 8 mutants (γ-irradiated) were examined

• 6 fragments of vestigial gene were analyzed• More than 60 polymerase chain reactions were carried

out• As you can see from the results of PCR only 2 mutants

have one deletion of different fragments each

Conclusions

• Such a little quantity of PCR-detected deletions could be caused by specific action of γ-rays. Because the type of radiation mentioned above induce point damage (as a rule) that cannot be detected by PCR

• In our research work only exones were examined. But there is a probability to discover deletions in intrones. So it can be the next step in analysis of this mutants

Structure of the cinnabar gene

Visualization of Electrophoresis Using UV Light

PCR results of the 1st,2nd and 4th fragments of the cinnabar gene

№ Dose (Gy) № of mutations

Fragments1st 2nd 4th

- Magarach + + +- Gomel + + +- CnS001 + + +- CnS003 + + +

1 5 γ Cn120 - + +2 10 γ Cn116 + - +3 20 γ Cn118 + + +4 40 γ Cn11 - + +5 40 γ Cn 16† - + +6 40 γ Cn20 - - -7 40 γ Cn40 - - -8 40 γ Cn48 + + +9 40 γ Cn50 + + +10 40 γ Cn57 - - -11 40 γ Cn65 - - -12 40 γ Cn86 + + +13 40 γ Cn93 + + +14 40 x Cn 98 + + +15 10 n (0.85) Cn46† - + +16 28 n +γ (Cf) Cn81 - - -17 28 n +γ (Cf) Cn82 - - -

6 out of 17(35%) irradiated cinnabar mutants show negative PCR for all three fragments studied.4 out of 11 (36%) remained show negative PCR for the 1st fragmentOnly 1 out of 11 (9%) showed negative PCR for the 2nd fragment.There were no negative PCR observed for the 11 that remained in the 4th fragment.

Conclusions

High frequency of the loss of the entire gene could be determined by the size and position

of the cinnabar gene on the chromosome

The beginning of the gene could be a hot spot for the irradiation for both gamma- and

neutron radiation

General Conclusions

• We have studied the molecular alterations induced by ionizing radiation at 2 Drosophila genes with different sizes, structure and position on the chromosomes.

• Two different genes-targets react in different ways on the action of radiation. They have different picture of radiomutability.

• It is suggested that this differences could be connected with different position of 2 genes on the chromosome.

Thanks for your attention!