The Antitumor Efficacy of a Complex Based on Two-Vector System for Coexpression of the Suicide Gene Fcu1 and Cre Recombinase.

In this study, we evaluated the antitumor activity of a gene therapy complex in which the tumor-specific control of the expression of the effector suicide gene FCU1 was performed using a two-vector system based on the site-specific Cre-LoxP recombinase system. The complex of interest showed a high therapeutic potential in a mouse colon adenocarcinoma model.

[Affinity capture of specific DNA fragments with the use of short synthetic sequences].

The ability of short peptide nucleic acid (PNA) oligomers and oligonucleotides containing modified residues of 5-methylcitidine, 2-aminoadenosine and 5-propynyl-2'-deoxyuridine (strong binding oligonucleotides, SBO) to affinity capture the target double-stranded DNA fragment from mixture by means of the end invasion was compared. Both types of probes were highly effective at the conditions used. The SBO-based probes may represent a handy and easily prepared alternative to PNA for selection of target DNA fragments from mixtures.

[Genome similarity of Baikal omul and sig].

Two members of the Baikal sig family, a lake sig (Coregonus lavaretus baicalensis Dybovsky) and omul (C. autumnalis migratorius Georgi), are close relatives that diverged from the same ancestor 10-20 thousand years ago. In this work, we studied genomic polymorphism of these two fish species. The method of subtraction hybridization (SH) did not reveal the presence of extended sequences in the sig genome and their absence in the omul genome. All the fragments found by SH corresponded to polymorphous noncoding genome regions varying in mononucleotide substitutions and short deletions. Many of them are mapped close to genes of the immune system and have regions identical to the Tc-1-like transposons abundant among fish, whose transcription activity may affect the expression of adjacent genes. Thus, we showed for the first time that genetic differences between Baikal sig family members are extremely small and cannot be revealed by the SH method. This is another endorsement of the hypothesis on the close relationship between Baikal sig and omul and their evolutionarily recent divergence from a common ancestor.

SSCP screening of individual aptamers.

Aptamers are specific binding nucleic acids that emerge from in vitro selection. During the systematic evolution of ligands by exponential enrichment (SELEX) procedure, analysis of the sequences of the numerous selected individual molecules becomes an important step in the final stage of aptamer selection. The sequencing of cloned aptamers from the selected pool generally reveals groups of identical sequences and rarely occurring individual aptamers. This study demonstrates an approach similar to the single strand conformation polymorphism (SSCP) method used for mutation testing in genes. Human angiotensin I-specific aptamers have been used to show the efficiency of the SSCP method to classify selected individual sequences into identity groups, which minimizes sequencing efforts. Additionally this approach allows the rapid isolation and identification of aptamers from a mixture.

Determination of the patterns of hexanucleotide distribution along DNA by electron microscopy.

Hexanucleotides containing modified bases (5-methylcytosine and 2-aminoadenine instead of cytosine and adenine) with increased capacities to bind complementary DNA sequences were used to map the distribution of their complementary sequences in a DNA target using electron microscopy. The method used hexamers to initiate DNA polymerase directed DNA synthesis at complementary sequences along a template. DNA synthesis was limited to about 200 residues by using a low concentration of deoxynucleotide precursors. During DNA synthesis a biotin ligand was incorporated to facilitate the subsequent binding of an electron-dense label (streptavidin-labeled colloidal gold particles) into newly synthesized DNA chains. The method can be implemented with commercially available products. The results demonstrate that the approach can be used to compare primary structural features of DNA fragments. The principles of the method can be adapted to a variety of single molecule detection methods such as electron, scanning tunneling, or atomic force microscopies.

Detection of planarian Antennapedia-like homeobox genes expressed during regeneration.

Using the polymerase chain reaction with degenerate oligo primers we identified six new Antennapedia-like homeobox sequences expressed in a regenerating planarian. cDNA fragments containing the entire homeobox sequences for four of these genes were obtained. Two of them, Dutarh-3 and Dutarh-4, belong to the classical Antennapedia type and display extensive identity with Antennapedia- and Deformed-type homeodomains respectively. The third homeodomain, Dutarh-1, exhibits some similarity to the Hox1.5-, AbdB- and Dfd-type homeodomains and a fourth gene, Dutarh-6, is very similar to Mox2 in the mouse. Our results suggest that, in spite of their simple body plan, planarians contain a number of Antennapedia-like homeobox genes, probably enough to fill a cluster such as found in higher animals.

[The cloning of fragments of homeobox genes expressed during planarian regeneration]. / Klonirovanie fragmentov gomeobokssoderzhashchikh genov, ékspressiruiushchikhsia v khode regeneratsii planarii.

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.

Substitution of 2-aminoadenine and 5-methylcytosine for adenine and cytosine in hybridization probes increases the sensitivity of DNA fingerprinting.

An oligonucleotide (m5C-am2A-m5C)5 containing 2'-amino-deoxyadenosine (am2A) and 5-methyldeoxycytidine (m5C) residues has been synthesized and compared with unsubstituted pentadecadeoxyribonucleotide (CAC)5 as a hybridization probe for DNA fingerprinting. It was shown that considerably higher sensitivity can be achieved with the modified analog.

[Intensification of genomic dactyloscopy by means of hybridization with modified oligonucleotides, forming duplexes with increased stability]. / Intensifikatsiia genomnoi daktiloskopii putem gibridizatsii s modifitsirovannymi oligonukleotidami, obrazuiushchimi dupleksy s povyshennoi stabil'nostiu.

The modified oligodeoxyribonucleotide (m5C-n2A-m5C)5 containing 5-methylcytosine and 2-aminoadenine instead of cytosine and adenine residues, respectively has been used as a hybridisation probe in DNA fingerprinting. The oligonucleotide, due to the substitutions forms more stable duplexes with complementary sequence in DNA than the corresponding nonmodified pentadecanucleotide. The comparison with its natural counterpart displays considerably increased intensity of bands in patterns obtained with modified analog. The use of such analogues can increase sensitivity and shorten time of DNA fingerprinting.

A method for isolation of sequences missing in one of two related genomes.

We describe a novel technique for isolation of sequences that are present in one genome (tracer), but absent in another (driver). Tracer DNA, cleaved with Sau 3A and capped with a single stranded PCR adapter, is allowed to hybridize with an excess of sheared biotinylated driver; biotinylated DNA and its hybrids with the tracer are removed by phenol/chloroform extraction after incubation with streptavidin. After several rounds of subtraction the ends of self-annealed tracer molecules from the nonextractable fraction are filled-in with Tag polymerase and amplified, using the single stranded PCR adapter as a primer. The method has been applied to purification of fragments from a 2.9 kb plasmid added to E. coli DNA at equimolar quantity. Plasmid derived fragments (250-1000 bp), initially comprising 1/1400th part of tracer DNA, were purified to homogeneity after two rounds of subtraction followed by PCR.

Interaction of ribo- and deoxyriboanalogs of yeast tRNA(Phe) anticodon arm with programmed small ribosomal subunits of Escherichia coli and rabbit liver.

A synthetic ribooligonucleotide, r(CCAGACUGm-AAGAUCUGG), corresponding to the unmodified yeast tRNA(Phe) anticodon arm is shown to bind to poly(U) programmed small ribosomal subunits of both E. coli and rabbit liver with affinity two order less than that of a natural anticodon arm. Its deoxyriboanalogs d(CCAGACTGAAGATCTGG) and d(CCAGA)r(CUGm-AAGA)d(TCTGG), are used to study the influence of sugar-phosphate modification on the interaction of tRNA with programmed small ribosomal subunits. The deoxyribooligonucleotide is shown to adopt a hairpin structure. Nevertheless, as well as oligonucleotide with deoxyriboses in stem region, it is not able to bind to 30S or 40S ribosomal subunits in the presence of ribo-(poly(U] or deoxyribo-(poly (dT) template. The deoxyribooligonucleotide also has no inhibitory effect on tRNA(Phe) binding to 30S ribosomes at 10-fold excess over tRNA. Neomycin does not influence binding of tRNA anticodon arm analogs used. Complete tRNA molecule and natural modifications of anticodon arm are considered to stabilize the arm structure needed for its interaction with a programmed ribosome.

Distinct modes of poliovirus polyprotein initiation in vitro.

RNAs of poliovirus type 1 and type 3 were translated in extracts from Krebs-2 cells after annealing with oligodeoxyribonucleotides complementary to different sites in the 5'-untranslated region (5'-UTR). Due to a high level of endogenous RNase H activity in the extracts, such RNAs appeared to be efficiently 5'-truncated prior to translation. The observed levels of initiation on differently truncated templates suggested that a region in the middle of the poliovirus 5'-UTR is essential for the cap-independent initiation of viral polyprotein synthesis. The data reported here, in conjunction with the results from other laboratories, permitted to relate the essential cis-acting control elements to the 5'-UTR secondary structure domains defined previously (E.V. Pilipenko et al., Virology 168, 201-209). However, the removal of these domains activated another mode of polyprotein initiation, which appeared to require another set of translation initiation factors.

[Synthesis and properties of DNA duplexes containing hydrocarbon bridges instead of a nucleoside residue]. / Sintez i svoistva DNK-dupleksov, soderzhashchikh uglevodorodnye mostiki vmesto odnogo iz nukleozidnykh ostatkov.

DNA duplexes 14 bp long containing an EcoRII and MvaI restriction site in which a nucleoside is substituted by 1,3-diaminopropane or 1,3-propanediol residue have been chemically synthesized. Diaminopropane bridge was introduced by the chemical ligation, whereas the oligonucleotide containing propanediol was prepared by automatic solid phase phosphoroamidite method on "Victoria-4M" synthesizer. As CD and UV spectra show, the modification destabilises the duplex by 18-20 degrees C without essential distortion of the double helix, except for increase of the conformational mobility in the modified site.

[Automated synthesis of oligodeoxyribonucleotides with terminal phosphate groups]. / Avtomticheskii sintez oligodezoksiribonukleotidov s kontsevymi fosfatnymi gruppami.

3'- and 5'-phosphorylated oligodeoxyribonucleotides have been synthesized on the "Victoria-4M" automatic synthesizer by phosphoramidite method. Two approaches have been suggested: introduction of a terminal ribo-unit as a potential source of phosphate group or the use of hydroxyl-containing polymer supports which loose the end product of the synthesis via beta-elimination reaction. Yields of oligonucleotides obtained according to both schemes proved to be almost identical. Using the first approach, oligonucleotides containing units with altered configuration of the sugar (xylo-thymidine and arabino-uridine) have been obtained.

Interaction of EcoRII restriction and modification enzymes with synthetic DNA fragments. V. Study of single-strand cleavages.

Concatemer DNA duplexes which contain at the EcoRII restriction endonuclease cleavage sites (formula; see text) phosphodiester, phosphoamide or pyrophosphate internucleotide bonds have been synthesized. It has been shown that this enzyme did not cleave the substrate at phosphoamide bond. EcoRII endonuclease catalyzes single-strand cleavages both in dA- and dT-containing strands of the recognition site if the cleavage of the other strand has been blocked by modification of scissile bond or if the other strand has been cleaved. This enzyme interacts with both strands of the DNA recognition site, each of them being cleaved independently on the cleavage of another one. Nucleotide sequences flanking the EcoRII site on both sides are necessary for effective cleavage of the substrate.