P A Zhulidov

Russian Academy of Sciences, Moskva, Moscow, Russia

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Publications (12)33.7 Total impact

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    ABSTRACT: In a cellular transcriptome, the number of mRNA copies per gene may differ by several orders. In cDNA libraries, performed from mRNA, these proportions are the same. Normalization methods allow us to equalize numbers of gene’s copies in the library. Normalized cDNA libraries are used to discover new genes transcribed at relatively low levels or for functional screenings. Here, we observed different cDNA libraries normalization methods, which were based on hybridization (renaturation) of cDNA or DNA, or RNA. Also we described duplex-specific nuclease (DSN) normalization protocol – simple and effective cDNA libraries normalization method.
    06/2007: pages 97-124;
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    ABSTRACT: We overexpressed duplex-specific nuclease (DSN) from Kamchatka crab in Escherichia coli cells and developed procedures for purification, renaturation, and activation of this protein. We demonstrated identity of the properties of the native and recombinant DSN. We also successfully applied the recombinant DSN for full-length cDNA library normalization.
    Biochemistry (Moscow) 06/2006; 71(5):513-9. · 1.15 Impact Factor
  • EJC Supplements 06/2006; 4(6):35-35. · 2.71 Impact Factor
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    ABSTRACT: We developed a new method for the preparation of normalized cDNA libraries enriched with full-length sequences. It is based on the properties of the recently characterized duplex-specific nuclease from the hepatopancreas of the Kamchatka crab. The duplex-specific nuclease is thermostable, it effectively cleaves double-stranded DNA and is inactive toward single-stranded DNA (Shagin et al., Genome Res., 2002, vol. 12, pp. 1935-1942). Our method enables the normalization of cDNA samples enriched with full-length sequences without use of laborious and ineffective stages of physical separation. The efficiency of the method was demonstrated in model experiments using cDNA samples from several human tissues.
    Bioorganicheskaia khimiia 01/2005; 31(2):186-94.
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    ABSTRACT: A new modification of the single nucleotide polymorphism (SNP) analysis (DSNP, duplex-specific nuclease preference) method using the duplex-specific nuclease from the king crab was proposed. The method was used to study SNPs in the following human genes: kRAS, nRAS, hRAS, and p53, the genes of blood coagulation factor V, methyltetrahydrofolate reductase, prothrombin, and apolipoprotein E and a deletion in the BRCA1 gene. DSNP was shown to be useful for the estimation of the mutant allele content in DNA samples. A system for the simultaneous identification of several adjacent single-nucleotide polymorphisms in the kRAS gene was proposed. The approaches could be used to develop test systems for the detection of SNPs in human genes. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 6; see also http://www.maik.ru.
    Bioorganicheskaia khimiia 01/2005; 31(6):627-36.
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    ABSTRACT: A new modification of the single nucleotide polymorphism (SNP) analysis (DSNP, duplex-specific nuclease preference) method using the duplex-specific nuclease from the king crab was proposed. The method was used to study SNPs in the following human genes: kRAS, nRAS, hRAS, and p53, the genes of blood coagulation factor V, methyltetrahydrofolate reductase, prothrombin, and apolipoprotein E and a deletion in the BRCA1 gene. DSNP was shown to be useful for the estimation of the mutant allele content in DNA samples. A system for the simultaneous identification of several adjacent single-nucleotide polymorphisms in the kRAS gene was proposed. The approaches could be used to develop test systems for the detection of SNPs in human genes.
    Russian Journal of Bioorganic Chemistry 01/2005; 31(6):567-575. · 0.52 Impact Factor
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    ABSTRACT: We developed a novel simple cDNA normalization method [termed duplex-specific nuclease (DSN) normalization] that may be effectively used for samples enriched with full-length cDNA sequences. DSN normalization involves the denaturation-reassociation of cDNA, degradation of the double-stranded (ds) fraction formed by abundant transcripts and PCR amplification of the equalized single-stranded (ss) DNA fraction. The key element of this method is the degradation of the ds fraction formed during reassociation of cDNA using the kamchatka crab DSN, as described recently. This thermostable enzyme displays a strong preference for cleaving ds DNA and DNA in DNA-RNA hybrid duplexes compared with ss DNA and RNA, irrespective of sequence length. We developed normalization protocols for both first-strand cDNA [when poly(A)+ RNA is available] and amplified cDNA (when only total RNA can be obtained). Both protocols were evaluated in model experiments using human skeletal muscle cDNA. We also employed DSN normalization to normalize cDNA from nervous tissues of the marine mollusc Aplysia californica (a popular model organism in neuroscience) to illustrate further the efficiency of the normalization technique.
    Nucleic Acids Research 02/2004; 32(3):e37. · 8.81 Impact Factor
  • EJC Supplements 02/2004; 2(1):41-41. · 2.71 Impact Factor
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    ABSTRACT: We have characterized a novel nuclease from the Kamchatka crab, designated duplex-specific nuclease (DSN). DSN displays a strong preference for cleaving double-stranded DNA and DNA in DNA-RNA hybrid duplexes, compared to single-stranded DNA. Moreover, the cleavage rate of short, perfectly matched DNA duplexes by this enzyme is essentially higher than that for nonperfectly matched duplexes of the same length. Thus, DSN differentiates between one-nucleotide variations in DNA. We developed a novel assay for single nucleotide polymorphism (SNP) detection based on this unique property, termed "duplex-specific nuclease preference" (DSNP). In this innovative assay, the DNA region containing the SNP site is amplified and the PCR product mixed with signal probes (FRET-labeled short sequence-specific oligonucleotides) and DSN. During incubation, only perfectly matched duplexes between the DNA template and signal probe are cleaved by DSN to generate sequence-specific fluorescence. The use of FRET-labeled signal probes coupled with the specificity of DSN presents a simple and efficient method for detecting SNPs. We have employed the DSNP assay for the typing of SNPs in methyltetrahydrofolate reductase, prothrombin and p53 genes on homozygous and heterozygous genomic DNA.
    Genome Research 01/2003; 12(12):1935-42. · 14.40 Impact Factor
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    ABSTRACT: A technique for the Selective Differential Display of RNAs containing Interspersed Repeats (SDDIR) has been elaborated. SDDIR involves two main steps: (1) selective amplification by RT-PCR of a subset of the total cellular RNA containing a certain type of repetitive element, and (2) side-by-side display of the amplicons derived from the tissues under comparison by means of gel electrophoresis in parallel lanes. The technique was used to compare the expression of transcripts containing LTR (Long Terminal Repeat) sequences derived from human endogenous retrovirus K (HERV-K) in testicular germ cell tumors and in corresponding normal tissue. SDDIR enabled us to obtain an overview of LTRs represented in the total transcribed fraction and to reveal differences in transcription patterns of the LTRs in normal and tumor tissues. An unexpectedly large number of LTRs was found to be transcribed, and the levels of many of the transcripts differed between normal and tumor tissues.
    Molecular Genetics and Genomics 02/2002; 266(5):796-805. · 2.88 Impact Factor
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    ABSTRACT: A transcript containing the long terminal repeat (LTR) and the sequence homologous to the KIAA1245 mRNA fragment were revealed among the transcribed LTRs of human endogenous retroviruses of the K family in normal and tumor tissues. Ten other sequences with a high level of homology to the KIAA1245 mRNA were found in the GenBank. The intron–exon structures were determined for all the sequences, and their exon sequences were compared. The comparison showed that they differ both in the extent of their exon homology and in the presence or absence of the HERV-K LTR in the third intron. The revealed sequences form a new gene family that comprises at least four subfamilies. Two of these subfamilies have the LTR, and the other two do not. We showed by PCR that the LTR was integrated into the introns after the divergence of the orangutan evolutionary branch from other hominoids but before the divergence of the gorilla branch, i.e., 8–13 million years ago.
    Russian Journal of Bioorganic Chemistry 01/2002; 28(4):312-315. · 0.52 Impact Factor
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    ABSTRACT: A transcript containing the long terminal repeat (LTR) and the sequence homologous to the KIAA1245 mRNA fragment were revealed among the transcribed LTRs of human endogenous viruses of the K family in normal and tumor tissues. Ten other sequences with a high level of homology to the KIAA1245 mRNA were found in the GenBank. The intron-exon structures were determined for all the sequences, and their exon sequences were compared. The comparison showed that they differ both in the extent of the exon homology and in the presence or absence of the HERV-K LTR in the third intron. The revealed sequences form a new gene family that comprises at least four subfamilies. Two of these subfamilies have the LTR, and the other two do not. We showed by PCR that the LTR was integrated into the introns after the divergence of the orangutan evolutionary branch from other hominoids but before the divergence of the gorilla branch, i.e., 8-13 million years ago. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2002, vol. 28, no. 4; see also http://www.maik.ru.
    Bioorganicheskaia khimiia 01/2002; 28(4):346-50.