1. PCR assay of the molecular alternations at cinnabar and vestigial genes of Drosophila melanogaster after γ-rays and neutron action
Igor D. Alexandrov, Ph. D., Dr. Sci. (Biology), chief. Sci. res.
Margarita V. Alexandrova, Ph. D., senior sci. res.
Aleksievich Olga
Sheresh Irina
Lebogang Sepini

2. 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

3. Potential genetic risks
Nuclear explosion
Technogenic catastrophes
Sun radiation

4. 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

5. Vestigial mutant
Cinnabar mutant
Wild type
Chromosome map

6. Main steps
Isolation of DNA
Polymerase chain reaction
Electrophoresis

7. Isolation of DNA
DNAs 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™.

8. 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)

9. Amplificator

10. Gel electrophoresis

11. Scheme of vestigial gene

12. Ex 6-8
Ex 3
Ex 5
Ex 4
Ex 1
Ex 2

13. 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
307 + 2
308
vg85e4
γ, 20 4
vg84f
X, 40 - 5
vg76d1
γ, 40 6
vg78b1 7
vg78b3
vg76d2 9
vg78a2
vg85d1
n, 15

14. 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

15. 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

16. Structure of the cinnabar gene

17. Visualization of Electrophoresis Using UV Light

18. PCR results of the 1st,2nd and 4th fragments of the cinnabar gene
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 fragment
Only 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. №
Dose (Gy)
№ of mutations
Fragments
1st
2nd
4th -
Magarach +
Gomel
CnS001
CnS003 1
5 γ
Cn120 2
10 γ
Cn116 3
20 γ
Cn118 4
40 γ
Cn11 5
Cn 16† 6
Cn20 7
Cn40 8
Cn48 9
Cn50 10
Cn57 11
Cn65 12
Cn86 13
Cn93 14
40 x
Cn 98 15
10 n (0.85)
Cn46† 16
28 n +γ (Cf)
Cn81 17
28 n +γ (Cf)
Cn82

19. 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

20. 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.

21. Thanks for your attention!