Laboratory of Structure of Genomes
Head G.M.Dymshits, Dr.Biol.Sci., Prof.
Localisation of the somatomammotropin gene on human chromosome 17
Scanning of capron after the appearance of ligation products hybridizing with the DNA template immobilized by UV-radiation
In situ hybridization of a mixture containing mink and mouse cells with tetramethylrhodamine labeled mouse DNA (yellow) and fluoresceine labeled mink DNA (green)
The amplification strategy. A schematic localisation of P1, P2, and P3, primers and the size of amplification products specific to the normal (N) and "mutant" (S) atpA genes from the sugar beet mitochondrial genome. H - the cleavage site for restriction endonucleases HindIII; B - the cleavage site for restriction endonuclease BamHI.
A new method of DNA and RNA chemical modification was developed. It allows to introduce biotin, different fluorochromes or other nonradioactive signal compounds into polynucleotide probes. At the first step of DNA modification, the aminooxy group reacts selectively with cytosine residue, at the second step, the reporter compounds attach to aminogroup introduced. The labeling procedure is simple and allows rapid (in several hours) production of sufficient amounts (up to several milligrams) of nonradioactive labeled probes. The detection sensitivity of probes, which were biotinylated as described, with streptavidin-alkaline phosphatase conjugate amounts to 0.5- 0.2 pg of target DNA.
The application of nonradioactive labeled probes for in situ hybridization allows to map mammalian chromosomes with accuracy not attainable by radioautography. Increase in the spatial resolution is very important for localization of unique nucleotide sequences. Using biotinylated probe the gene for somatomammotropin was localised on human metaphase chromosomes with the precision of one G-band in the 17q22 region .
The flexible labeling and detection system suggested makes it possible to reveal biotinylated probes not only colorimetrically, but also with the luminescent microscope, using fluorochromed avidin or antibodies to avidin carrying fluorescent labels.
The method of direct fluorescent in situ hybridization (DFISH) combines not only high precision and detailed resolution, but also allows bypassing tedious procedures of biochemical revealing with the use of enzymatic or fluorescent conjugates. The important advantage of DFISH is the possibility of the simultaneous use of hybridization probes carrying different fluorochromes. Multicolor probe detection using the fluorescent microscope allowing to increse sufficiently the spatial resolution and accelerate results. In 1986, for the first time in the world multicolor DFISH was performed at the Laboratory.
Using UV illumination as the universal way of DNA-target immobilization on membrane filters ("The way of hybridization of deoxyribonucleic acids". Patent 1 1640995 08.12.1990) and the flexible method of introducing nonradioactive label into the probe ("The way of producing nonradioactive labeled probe for revealing nucleotide sequences". Patent 1 2049821 10.12.1995), the highly-sensitive tests for revealing bovine leucosis provirus, malaria plasmodium in human blood and mycoplasmal contaminations of cell cultures were developed.
To diagnose hereditary diseases caused by point mutations, the colorimetric test system, based on ligation of tandem of short oligonucleotides was developed. The system allows in 2-3 hours to determine correctly and with high sensitivity not only the location, but also the type of a single-base substitution in any amplified region of genomic DNA.
As a result of ligation of tandem pN8+pN4++pN'8+Bio on template DNA under analysis in the solution the 20mer pN20+Bio forms. To discriminate two types of biotinylated oligonucleotides (pN20Bio and the component of a tandem pN'8Bio), the method of UV-immobilization of long DNA molecules on nylon was applied. After ligation and placing the reaction mixture onto the nylon only the longer product can be immobilized under UV illumination. The long template, in turn, holds the 20mer pN20Bio back on membrane due to the formalion of stable complex, whereas the octamer pN'8Bio), , forming a much less stable complex, is easily removed during washing steps.
The possibility of revealing point mutations in genomic DNA was examined on model systems with all possible single-base substitutions in tetramer sequence. In all cases, the 135 b.p. fragment of HIV1 DNA was used as a template. Ligation of 13 tandems was performed, 12 of which had all the possible mismatches in the tetramer _ DNa complex. The intense coloring was observed only in the case of ligation of tandem containing pCAGC tetranucleotide fully complementary to DNA fragment under analysis. When using tetramers having single-base substitution, the coloring intensity was always sufficiently lower than in perfect complex, though depending on the type of a mismatch.
Investigation of sugar beet mitochondrial genome structure and its rearrangements, associated with the CMS trait is carried out. A test system based on PCR with the use of three primers was developed, which allows to screen sugar beet plants by the type of their cytoplasm without taking phenotype into account. The three- primer system is adapted to the sequences of two genes, atpA and atp6, which are present only in the mitochondrial genome among all higher plants.
After the amplification reaction, either 2 reaction products should appear in the case the mitochondrial populations are heteroplasmatic or only one amplification product specific to the homoplasmatic mitochondrial population of either the pollen-sterile or the pollen-fertile plant.
Using the described system, we demonstrated for the first time that sugar beet plants spontaneously converted to the GMS trait are heteroplasmatic for the relative content of normal [N] or mutant (S) sequences of the atpA and atp6. genes. It was established that the heteroplasmic state of the mitochondrial intracellular population may be represented not only by the full S- or N-plasmotype, but also by sequences of the individual mitochondrial genes.