PubMed Health⌕ Search

Biomedical subjects

S Lukyanov

Publications and source records attributed to S Lukyanov.

18 recordsLinked to original sources
PubMed2004

Magnetic moment of the fragmentation-aligned 61Fe (9/2(+)) isomer.

We report on the g factor measurement of an isomer in the neutron-rich (61)(26)Fe (E(*)=861 keV and T(1/2)=239(5) ns). The isomer was produced and spin aligned via a projectile-fragmentation reaction at intermediate energy, the time dependent perturbed angular distribution method being used for the measurement of the g factor. For the first time, due to significant improvements of the experimental technique, an appreciable residual alignment of the nuclear spin ensemble has been observed, allowing a precise determination of its g factor, including the sign: g=-0.229(2). In this way we open the possibility to study moments of very neutron-rich short-lived isomers, not accessible via other production and spin-orientation methods.

Journal Article↗
PubMed2000

"Fluorescent timer": protein that changes color with time.

We generated a mutant of the red fluorescent protein drFP583. The mutant (E5) changes its fluorescence from green to red over time. The rate of color conversion is independent of protein concentration and therefore can be used to trace time-dependent expression. We used in vivo labeling with E5 to measure expression from the heat shock-dependent promoter in Caenorhabditis elegans and from the Otx-2 promoter in developing Xenopus embryos. Thus, E5 is a "fluorescent timer" that can be used to monitor both activation and down-regulation of target promoters on the whole-organism scale.

Animals↗
PubMed2000

Expression of PTTG and prc1 genes during telencephalic neurogenesis.

We present the first time/space analysis using in situ hybridization for PTTG and prc1 genes during development of the mouse telencephalon. During the stages E11.5-E13.5 PTTG and prc1 are expressed in most tissues of the embryo. Within the telencephalon, PTTG and prc1 are found exclusively inside of the ventricular zone (VZ). The intensity of the expression of both genes in the ventricular zone reaches its peak by E15.5. Expression starts to decrease by E18.5, it is still visible at least up to P2 and not detectable in the adult brains. Expression of the prc1 gene, but not that of the PTTG, is also found in the mitoticaly active cells outside of the VZ within the telencephalon. Most of the cells expressing the PTTG gene were found in the lower part of the ventricular zone suggesting that the level of PTTG mRNA is regulated during different phases of the mitotic cycle.

Animals↗
PubMed1999

Amplification of cDNA ends based on template-switching effect and step-out PCR.

A new method for amplifying cDNA ends is described which requires only first-strand cDNA synthesis and a single PCR to generate a correct product with very low or no background. The method can be successfully applied to total RNA as well as poly A+ RNA. The same first-strand cDNA can be used to amplify flanking sequences of any cDNA species present in the sample.

DNA, Complementary↗
PubMed1999

Suppression subtractive hybridization: a versatile method for identifying differentially expressed genes.

A new and highly effective method, termed suppression subtractive hybridization (SSH), has been developed for the generation of subtracted cDNA libraries. It is based primarily on a technique called suppression PCR, and combines normalization and subtraction in a single procedure. The normalization step equalizes the abundance of cDNAs within the target population and the subtraction step excludes the common sequences between the target and driver populations. As a result only one round of subtractive hybridization is needed and the subtracted library is normalized in terms of abundance of different cDNAs. It dramatically increases the probability of obtaining low-abundance differentially expressed cDNA and simplifies analysis of the subtracted library. The SSH technique is applicable to many molecular genetic and positional cloning studies for the identification of disease, developmental, tissue-specific, or other differentially expressed genes. This chapter provides detailed protocols for the generation of subtracted cDNA and differential screening of subtracted cDNA libraries. As a representative example we demonstrate the usefulness of the method by constructing a testis-specific cDNA library as well as using the subtracted cDNA mixture as a hybridization probe. Finally, we discuss the characteristics of subtracted libraries, the nature and level of background nondifferentially expressed clones in the libraries, as well as a procedure for the rapid identification of truly differentially expressed cDNA clones.

Animals↗
PubMed1999

PCR based targeted genomic and cDNA differential display.

We previously described a targeted genomic differential display method (TGDD: Broude NE, Chandra A, Smith CL. Differential display of genomic subsets containing specific interspersed repeats. Proc. Natl. Acad. Sci. USA 1997;94:4548-53). In that method, presently characterized as method I, targeting was accomplished by capturing DNA fragments containing specific a sequence by hybridization with complementary single-stranded DNA. The captured fragments were amplified by PCR. Here, we describe method II where targeting is accomplished by PCR using primers specific to the target sequence. Method II takes advantage of PCR suppression to eliminate fragments not containing the target sequence (Siebert PDA, Chenchik A, Kellogg DE, Lukyanov KA and Lukyanov SA. An improved PCR method for walking in uncloned genomic DNA. Nucleic Acids Res 1995;23:1087-1088). Targeting focuses analysis on and around interesting areas and additionally serves to reduce the complexity of the amplified subset. These approaches are useful to amplify genome subsets containing a variety of targets including various conserved sequences coding for cis-acting elements or protein motifs.

Alleles↗
PubMed1998

Inductive interactions regulating body patterning in planarian, revealed by analysis of expression of novel gene scarf.

Subtractive hybridization was used to search for the early difference in gene expression between anteriorly and posteriorly regenerating tissues of the same region of the planarian body. A sequence (named scarf) specific for posteriorly regenerating tissue was isolated, encoding a soluble C-type lectin consisting of two slightly different carbohydrate-recognition domains. Such an unusual bivalent structure allows attribution of the protein to a novel subfamily of C-type lectins. scarf expression in intact planarian is rather abundant and follows a characteristic pattern not linked to any known morphological structure. We performed a series of experiments using scarf as a molecular marker. Its expression was monitored during different types of regeneration by whole-mount in situ hybridization and reverse-transcription polymerase chain reaction. The obtained data suggest that scarf expression is positively regulated by anterior tissues closely adjacent to the scarf-expressing region, so that their surgical removal results in rapid scarf switch-off. In turn, tissues expressing scarf seem to inhibit its activation anteriorly. This indicates that at least some of the body patterning events in planarians are based on a system of reciprocal inductive interactions rather than on a global morphogen gradient.

Amino Acid Sequence↗
PubMed1997

Ordered differential display: a simple method for systematic comparison of gene expression profiles.

A method for display of 3'-end restriction fragments of cDNAs is proposed, extending the idea reported recently. First, representative pools of such fragments are selectively amplified using PCR suppression effect. Then, simplified subsets of these fragments suitable for comparison by PAGE are amplified by adapter-specific primers extended by two randomly picked bases at their 3'ends. By testing all possible combinations of extended primers the whole mRNA pool may be systematically investigated. The method was applied to search for molecular regional markers of freshwater planarian Dugesia tigrina .

Animals↗
PubMed1997

Construction of cDNA libraries from small amounts of total RNA using the suppression PCR effect.

Here we describe a method for preparing high-quality cDNA libraries from total RNA. By this method, double-stranded (ds) cDNA ligated with a specially designed ds adaptor is amplified by PCR using a modified T-primer and another primer corresponding to the outer part of the adaptor. The suppression PCR effect strongly inhibits the amplification of poly(A) RNA, thereby reducing background. This method leads to amplification of high-quality cDNA, facilitating the construction of representative cDNA libraries from as little as 10-100 ng of total RNA.

DNA, Complementary↗
PubMed1996

Genes from the medicinal leech (Hirudo medicinalis) coding for unusual enzymes that specifically cleave endo-epsilon (gamma-Glu)-Lys isopeptide bonds and help to dissolve blood clots.

We previously detected in salivary gland secretions of the medicinal leech (Hirudo medicinalis) a novel enzymatic activity, endo-epsilon(gamma-Glu)-Lys isopeptidase, which cleaves isopeptide bonds formed by transglutaminase (Factor XIIIa) between glutamine gamma-carboxamide and the epsilon-amino group of lysine. Such isopeptide bonds, either within or between protein polypeptide chains are formed in many biological processes. However, before we started our work no enzymes were known to be capable of specifically splitting isopeptide bonds in proteins. The isopeptidase activity we detected was specific for isopeptide bonds. The enzyme was termed destabilase. Here we report the first purification of destabilase, part of its amino acid sequence isolation and sequencing of two related cDNAs derived from the gene family that encodes destabilase proteins, and the detection of isopeptidase activity encoded by one of these cDNAs cloned in a baculovirus expression vector. The deduced mature protein products of these cDNAs contain 115 and 116 amino acid residues, including 14 highly conserved Cys residues, and are formed from precursors containing specific leader peptides. No homologous sequences were found in public databases.

Amino Acid Sequence↗
PubMed1996

Enzyme from the medicinal leech (Hirudo medicinalis) that specifically splits endo-epsilon(-gamma-Glu)-Lys isopeptide bonds: cDNA cloning and protein primary structure.

Earlier we detected a novel enzymatic activity in salivary gland secretion of the medicinal leech, splitting isopeptide bonds between the glutamine gamma-carboxamide and lysine epsilon-amino group. This activity is due to destabilase. We described its partial amino acid sequence and sequences to two closely related cDNAs, but none of them perfectly matched the protein isolated. Here we report the isolation and sequence peculiarities of the third cDNA of the family as well as the complete sequence of the destabilase protein. The inferred mature protein product of this cDNA matches the independently determined destabilase protein sequence. It contains 115 amino acid residues including 14 highly conserved Cys residues and is formed from a precursor containing specific leader peptide.

Amino Acid Sequence↗
PubMed1996

Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries.

A new and highly effective method, termed suppression subtractive hybridization (SSH), has been developed for the generation of subtracted cDNA libraries. It is based primarily on a recently described technique called suppression PCR and combines normalization and subtraction in a single procedure. The normalization step equalizes the abundance of cDNAs within the target population and the subtraction step excludes the common sequences between the target and driver populations. In a model system, the SSH technique enriched for rare sequences over 1,000-fold in one round of subtractive hybridization. We demonstrate its usefulness by generating a testis-specific cDNA library and by using the subtracted cDNA mixture as a hybridization probe to identify homologous sequences in a human Y chromosome cosmid library. The human DNA inserts in the isolated cosmids were further confirmed to be expressed in a testis-specific manner. These results suggest that the SSH technique is applicable to many molecular genetic and positional cloning studies for the identification of disease, developmental, tissue-specific, or other differentially expressed genes.

Base Sequence↗
PubMed1996

New isomer 32Alm.

Explore the source record for details and available documents.

Journal Article↗
PubMed1996

Full-length cDNA cloning and determination of mRNA 5' and 3' ends by amplification of adaptor-ligated cDNA.

An efficient cDNA amplification procedure is described for determining of the 5' and 3' ends of mRNAs and cloning full-length cDNAs. In this approach, a double-stranded (ds) adaptor is ligated to both ends of a library of ds cDNA by T4 DNA ligase. This adaptor-ligated ds cDNA is then used to selectively amplify 5'- or 3'-cDNA fragments by PCR with a combination of gene-specific and adaptor-specific primers. This is a unified method for 5' and 3' rapid amplification of cDNA ends (RACE) from the same adaptor-ligated ds cDNA template. A specially designed adaptor combines features of "vectorette PCR" and "suppression PCR" technologies that significantly reduce background during amplification. The application of "long and accurate PCR" (LA PCR) technology makes possible the amplification of large RACE products and full-length cDNAs with high fidelity to the original mRNA. We investigated efficacy and limitations of this PCR-based approach for cDNA cloning by amplification of 5'- and 3'-RACE fragments and full-length cDNAs of three members of the abundant human actin gene family (1.3-1.9 kb), the medium abundance transferrin receptor mRNA (5.0 kb) and the low-medium abundance insulin-like growth factor II receptor mRNA (9.1 kb).

Actins↗