Biomedical subjects

Dmitry A Shagin

Publications and source records attributed to Dmitry A Shagin.

5 recordsLinked to original sources

PubMed2004

Simple cDNA normalization using kamchatka crab duplex-specific nuclease.

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.

Animals↗

PubMed2004

GFP-like proteins as ubiquitous metazoan superfamily: evolution of functional features and structural complexity.

Homologs of the green fluorescent protein (GFP), including the recently described GFP-like domains of certain extracellular matrix proteins in Bilaterian organisms, are remarkably similar at the protein structure level, yet they often perform totally unrelated functions, thereby warranting recognition as a superfamily. Here we describe diverse GFP-like proteins from previously undersampled and completely new sources, including hydromedusae and planktonic Copepoda. In hydromedusae, yellow and nonfluorescent purple proteins were found in addition to greens. Notably, the new yellow protein seems to follow exactly the same structural solution to achieving the yellow color of fluorescence as YFP, an engineered yellow-emitting mutant variant of GFP. The addition of these new sequences made it possible to resolve deep-level phylogenetic relationships within the superfamily. Fluorescence (most likely green) must have already existed in the common ancestor of Cnidaria and Bilateria, and therefore GFP-like proteins may be responsible for fluorescence and/or coloration in virtually any animal. At least 15 color diversification events can be inferred following the maximum parsimony principle in Cnidaria. Origination of red fluorescence and nonfluorescent purple-blue colors on several independent occasions provides a remarkable example of convergent evolution of complex features at the molecular level.

Animals↗

PubMed2002

Altering electrical connections in the nervous system of the pteropod mollusc Clione limacina by neuronal injections of gap junction mRNA.

Neurons can communicate with each other either via exchange of specific molecules at synapses or by direct electrical connections between the cytoplasm of either cell [for review see Bruzzone et al. (1996) Eur. J. Biochem., 238, 1-27]. Although electrical connections are abundant in many nervous systems, little is known about the mechanisms which govern the specificity of their formation. Recent cloning of the innexins--gap junction proteins responsible for electrical coupling in invertebrates (Phelan et al. (1998) Trends Genet., 14, 348-349], has made it possible to study the molecular mechanisms of patterning of the electrical connections between individual neurons in model systems. Here we demonstrate that intracellular injection of mRNA encoding the molluscan innexin Panx1 (Panchin et al. 2000 Curr. Biol., 10, R473-R474) drastically alters the specificity of electrical coupling between identified neurons of the pteropod mollusc Clione limacina.

Animals↗

PubMed2002

Identification and characterization of a new family of C-type lectin-like genes from planaria Girardia tigrina.

A novel family of C-type lectin-like genes, denoted multidomain free lectin (MDFL), was identified in the freshwater planaria Girardia (Dugesia) tigrina. We cloned several genes that encode proteins comprising a signal peptide and a number of consecutive C-type lectin-like domains (CTLDs) interconnected by short linker stretches. Analyses of genomic organization, CTLD amino acid sequences, and the overall architecture of these proteins indicate that planarian proteins are a separate family of C-type lectin-like proteins. These genes are expressed in specifically differentiated gland cells of planaria and the corresponding proteins are excreted as components of the planarian body surface mucus.

Amino Acid Motifs↗

PubMed2002

A novel method for SNP detection using a new duplex-specific nuclease from crab hepatopancreas.

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.

Animals↗