Butovskaya P.R1, Martirosyan I.A1, Baranov V.S2, Pavlova G.V1, Korochkin L.I1.
(1 - Institute of Gene Biology, RAS, Vavilova street 34/5, Moscow, 119334, Russia;e-mail: dna@biogen.msk.su)
In biological systems, mosaicism means the presence of two or more genetically different cell lines in an individual organism. It has been shown that somatic mosaicism is related to the development of many monogenic and mitochondrial diseases and cancer [1]. Significant progress in understanding the nature and mechanism of occurrence of somatic mosaicism has been reached in studies of hypervariable minisatellites and microsatellites of the human and animals [2, 3]. The high mutational variability of these loci may underlie the somatic mosaicism of an individual organism. Several minisatellite- and microsatellite-containing human loci that exhibit increased mutational activity have been revealed and studied using genetic engineering techniques [4-8]. Another approach that is used for revealing somatic mosaicism is multilocus DNA fingerprinting of individual organs and tissues. In this case, the relationship between genetically unstable loci with somatic mosaicism has been shown. This approach was first used to reveal microsatellite mutations in DNA of cancer tissues [9]. This is an universal approach for searching the most unstable genomic loci and cases of somatic mosaicism.
Materials and methods
DNA from the tissues and organs was isolated using standard phenol-chloroform technique with the use of proteinase K. DNA samples were treated with restriction endonucleases Bsu RI and Mva I and then subjected to blot hybridization analysis with 32P-labeled probes: oligonucleotides (GATA)4, (GACA)4, (CAC)5, and (GGCA)4.
Results
This was the first work to show somatic mosaicism in human at the microsatellite loci (GACA)n and (CAC)n using multilocus DNA fingerprinting.
Figure 1 and Figure 2 shows the (CAC) and (GACA) DNA fingerprinting of DNA samples from the tissues and organs from two different individuals. In both cases, we find difference in the DNA samples from the first individual (in the backbone - using (CAC)5 probe, and in the chorion - using (GACA)4 probe). The detection of mutant fragments only in the chorion DNA should be accounted for. It can be assumed that, similar to other mini-and microsatellites, different mechanisms (such as recombination, gene conversion, or replicational slipapage of DNA strands [3] determine the instability of the microsatellite-containing loci, causing mosaicism of cells, tissues, and organs in an individual. Possibly, the majority of mutations (as was shown for the locus Hm-2 [6]) occurs at the early stages of cell division. Then, during subsequent cell divisions, the rate of mutational process may change; as a result, one or another mutation, the number of cells for which in tissue is low, will not be detected by blot hybridization techniques. In addition, differential diminution of satellite DNA at the stage of development of blastogenic layers also cannot be ruled out. Development from the same layers may retain the complete set of repeating DNA sequences, whereas other cells lack them in the course of ontogeny. In this case, August Weisman, who assumed that cell differentiation is related to selective diminution of chromatin, was right [10].
In general, it can be assumed that the development of more sophisticated techniques for detecting mutant loci will show that somatic mosaicism is a ubiquitous phenomenon and that it is related to genetically determined processes of cell differentiation and biological development of a body.
Neurodegenerative desiases require a big social meaning. Development of cell technology, basically the study of stem cells during last ten years gives us a ways and possibilities for medical treatment of neirogenetic diseases. The transplantation of stem cells leads to recovery of central functions and may decrease the organic damage during modeling neurodegenerative processes in experimental animals. The intensification of regenerative processes is also shown using systematic and intracerebral introduction of bone marrow cells. Recent results allow us to hope on using cell therapy of bone marrow autologic cells. The ability of bone marrow stem cells to differentiate in neuronal direction is already known. Preliminary clinical proving of using bone marrow autologic stem cells by treating Parkinson s disease gave a positive result. However, the necessity of cells cultures certification is obvious. It is well known, that increasing the number of passages, which is necessary for culture growth, leads to genomic changes in culture cells, which could be quite dangerous for the patient. For the purpose of lowering the probability of getting the modified cell material in to the human organism we suggest to use as a test-system multilocus DNA-fingerprinting and locus-specific PCR. Following methods, allows to detect DNA changes and to control genomic transgressions of transplantants. Realization of genomic certification of transplantated cell culture could reduce the risk of getting the autologic cells with the changed DNA into patient organism.
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Figure captures
Figure A. DNA fingerprinting of organs and tissues of two human individuals with the use of (CAC)5 probe.
A) Lanes 1, 2, 3, 4, 5 and 6 show DNA isolated from liver, stomach, arm, backbone, brain and chorion respectively.
B) Lanes 7, 8, 9, 10, 11 and 12 show DNA isolated from heart, lungs, liver, adrenal glands, chorion and kidney respectively.
DNA of phage l treated with the restriction endonucieases Eco RI and Hind III was used as marker DNA fragments (kb).
Figure B. DNA fingerprinting of organs and tissues of two human individuals with the use of (GACA)4 probe.
A) Lanes 1, 2, 3, 4, 5 and 6 show DNA isolated from liver, stomach, arm, backbone, brain and chorion respectively.
B) Lanes 7, 8, 9, 10, 11 and 12 show DNA isolated from heart, lungs, liver, adrenal glands, chorion and kidney respectively.
DNA of phage l treated with the restriction endonucieases Eco RI and Hind III was used as marker DNA fragments (kb).
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