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Biomedical subjects

K A Luk'ianov

Publications and source records attributed to K A Luk'ianov.

10 recordsLinked to original sources
PubMed1999

[Selective suppression of polymerase chain reaction].

The selective suppression of the polymerase chain reaction and methods based upon it (construction of cDNA libraries from low amounts of biological material, subtractive hybridization and differential display of mRNA, fast cloning of full-size cDNA, chromosome walking, cloning in vitro, and others) are reviewed. These methods display a high effectiveness and, taken together, enable intricate DNA analyses to be performed--from the search for nontrivial sequences to the total sequencing of the corresponding genes.

3' Untranslated Regions↗
PubMed1997

[In vitro cloning of DNA fragments by one polymerase chain reaction].

An improved version of the in vitro DNA cloning method described earlier is proposed. The method allows amplification by polymerase chain reaction (PCR) of individual DNA molecules of an unknown primary structure followed by sequencing. The modifications described here provide for in vitro cloning by means of a 40-45-cycle PCR (the original protocol required two consecutive amplifications). In addition, the in vitro cloning is suggested to be carried out in special 96-well plates in the presence of ethidium bromide; upon UV irradiation, the wells containing amplified DNA fluoresce to make the analysis of all 96 wells unnecessary. The improved protocol makes the preparation of individual in vitro clones more straightforward and less expensive.

Base Sequence↗
PubMed1996

[Cloning cDNA for the ha-SDGF gene from a Syrian hamster cell line with increased metastatic potential using subtractive hybridization].

Using subtractive hybridization, a cDNA library containing over 50% of clones specific for a highly metastatic cell line was obtained from two hamster embryo fibroblast lines with different metastatic potentials. Most of the clones (83%) contained new sequences. One clone contained the ha-SDGF gene cDNA homologous to SDGF cDNA from rodents. The level of ha-SDGF mRNA expression was considerably higher in the highly metastatic cell line.

Amino Acid Sequence↗
PubMed1996

[A method for obtaining the normalized cDNA libraries based on the effect of suppression of polymerase chain reaction].

A new efficient method for obtaining cDNA libraries with equal representation of all cDNA types (equalized libraries) in a single round of equalization was developed. The method is based on differences in the renaturation kinetics of double-stranded cDNAs of different genes and allow the selection of the equalized single-stranded fraction resulted from the incomplete reassociation of the total cDNA without laborious and inefficient physical separation. The equalized single-stranded fractions are selectively amplified by polymerase chain reaction (PCR). The amplification of other DNA molecules is inhibited due to PCR suppression, i.e. the suppression of amplification of the DNA molecules flanked with long interval terminal repeats in PCR with a primer corresponding to the external moiety of the repeat. The efficiency of the developed method was estimated in obtaining an equalized cDNA library based on mRNA from the activated human T lymphocyte Jurkat cell line.

Base Sequence↗
PubMed1994

[Highly-effective subtractive hybridization of cDNA].

A scheme for subtractive hybridization is described allowing for a 500-1000 fold enrichment of low abundant cDNA. The scheme is based on the previously described principle of normalization of an initial mixture of differently represented cDNAs in the single-stranded portion of a tracer after the first round of subtraction and the principle of a trapper excluding the fraction of the double-stranded cDNAs formed during the first round from the subsequent PCR-amplification. The technique is simple and makes unnecessary the separation of the tracer, driver and hybrids formed after the subtraction.

Base Sequence↗
PubMedDate not supplied

[Key amino acid residues responsible for the color of green and yellow fluorescent proteins from the coral polyp Zoanthus].

Site-directed mutagenesis was used to study the structural basis of color diversity of fluorescent proteins by the example of two closely related proteins from one organism (coral polyp Zoanthus sp.), one of which produces green and the other, yellow fluorescence. As a result, the following conversions of emission colors were performed: from yellow to green, from yellow to a dual color (yellow and green), and from green to yellow. The saltatory character of the spectral transitions and the manifestation of the dual-color fluorescence suggest that chemically different fluorophores are responsible for the green and yellow fluorescence. The simultaneous presence of three residues, Gly63, Lys65, and Asp68, is necessary for the efficient formation of the yellow rather than green fluorophore. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2002, vol. 28, no. 4; see also http://www.maik.ru.

Amino Acid Sequence↗
PubMedDate not supplied

[A natural fluorescent protein that changes its fluorescence during maturation].

The gene of a new red fluorescent protein zoan2RFP from a coral polyp Zoanthus sp., a homologue of the known green fluorescent protein from the Aequorea victoria jellyfish, was cloned. At early maturation stages, zoan2RFP exhibits a green fluorescence, which then turns into the red one. A similar phenomenon was recently reported for the E5 mutant of the red fluorescent coral protein DsRed. Zoan2RFP differs from E5 by faster maturation kinetics and the complete disappearance of green fluorescence in the mature protein. Naturally occurring proteins of this type can be considered as intermediate forms between the green and red fluorescent proteins, which are formed during the microevolution of fluorescent proteins.

Amino Acid Sequence↗
PubMedDate not supplied

[The cloning of fragments of homeobox genes expressed during planarian regeneration].

The polymerase chain reaction with degenerate primers corresponding to the most conservative amino acids 16-21 (ELEKEF) and 49-54 (WFQNRR) of the Antennapedia class homeodomain was used for the amplification of cDNA from regenerating planarians (asexual race of Dugesia tigrina). A total of six new Antennapedia-like homeobox sequences, designated Dutarh-1-Dutarh-6 (Dugesia tigrina asexual race homeobox gene), were obtained. Their comparison with other homeobox genes using a "Genebee" software (the EMBL Data Library) showed that all sequences except Dutarh-6 belong to the Antennapedia class. Dutarh-6 is closely related to a recently described novel homeobox gene subfamily that includes mouse mesodermal homeobox genes Mox-1 and Mox-2 and rat homeobox gene Gax.

Amino Acid Sequence↗
PubMedDate not supplied

[An analysis of the expression of the genes containing the LeR-1 and VeR-1 sequences in the embryogenesis, regeneration and in the intact tissues of newts].

A method of construction of amplified cDNA libraries was developed on the basis of selective inhibition of cDNA amplification and modified for the studied models for analysis of expression of the genes containing LeR-1 and VeR-1 sequences. Time-related changes in expression of these genes were studied during regeneration of the adult lens and during embryogenesis of newts. The pattern of expression of the LeR-1 and VeR-1 genes proved to be not tissue-specific. A fragment adjoining 5'-area of the LeR-1 gene was obtained using a new approach: rapid amplification of cDNA ends by polymerase chain reaction (5'-RACE-PCR). Analysis of the LeR-1 clone primary structure using Gene Bank did not show essential homology with the known nucleotide sequences. Sequence LeR-1 is characterized by evolutionary conservation of genomic DNA in Pleurodeles waltlii, Xenopus laevis, and Rana temporaria.

Animals↗