[DNA Phenotyping of Remains from Elite Burials of the Khazar Period of Southern Russia].

Ancient DNA analyses help to solve the problems related to the genogeographic origin and migration patterns of populations. The Khazar Khaganate is a subject of controversy among researchers. Its complex historical development, lack of a sufficient number of artistic and written sources, the disappearance of representatives of Khazar culture leaves open the question of the appearance of the Khazars. DNA phenotyping of bone remains from elite burials of the Khazar period of Southern Russia was carried out with respect to eye color, hair color, skin color, and AB0 blood groups. Eight out of 10 individuals had brown eyes, dark hair (to varying degrees), and a predominantly dark skin during their lifetime. Individuals from two burials had gray-blue eyes, and one individual had blond hair. The most probable AB0 blood group was identified in eight people, of which five blood group 0 (I) group, four had blood group A (II), and one had blood group B (III). The allele frequency distribution was assessed for ten population-specific autosomal markers and suggested high heterogeneity for the ethnogeographic origin of the Khazars examined. The results are evidence for ethnocultural, genetic, and phenotypic diversity of the Khazar Khaganate.

[Universal Panel of Insertion/Deletion Polymorphisms and Biochip-Based Kit ChipID106 for Genetic Personal Identification].

A panel of 106 insertion/deletion (InDel) polymorphisms and a method of their genotyping on biochips were proposed as a new approach to genetic personal identification. Short lengths and low mutation rates are basic properties of InDel markers, which thus have significant advantages over short tandem repeats (STRs) widely used in forensics. The allele frequency distributions of all known InDel polymorphisms were studied in the five largest world populations (European, East Asian, South Asian, African, and American). Markers were selected to meet the following criteria: the minor allele frequency (MAF) is higher than 0.30; the physical distance between markers is greater than 3 Mb; there are no polymorphisms, tandem repeats, and palindromes in the flanking sequences; the AT/GC ratio is close to 1. A panel of 106 polymorphisms was thus formed; the average MAF was estimated at 0.396 in the five populations. The method developed for panel genotyping included one-step multiplex PCR and subsequent hybridization on a biological microarray. The average amplicon length was 72 bp. A sample of 201 residents of Moscow and St. Petersburg was tested to determine the main characteristics of the panel: the random matching probability (MP) was 1.89x 10^(-43) and the combined probability of paternity exclusion (CPE) was 0.99999999063. The method provides an alternative to molecular genetic personal identification based on the STR length variations.

[A Biochip for Genotyping Polymorphisms Associated with Eye, Hair, Skin Color, AB0 Blood Group, Sex, Y Chromosome Core Haplogroup, and Its Application to Study the Slavic Population].

This paper presents a method for genotyping a panel of 60 single nucleotide polymorphisms (SNPs) using single-stage PCR followed by hybridization on a hydrogel biochip. The pool of analyzed polymorphisms consists of 41 SNPs included in the HIrisPlex-S panel, 4 SNPs of the AB0 gene (261G>Del, 297A>G, 657C>T, 681G>A), markers of the AMELX and AMELY genes, and 14 SNP markers of the Y chromosome haplogroups: B (M60), C (M130), D (CTS3946), E (M5388), G (P257), H (M2920), I (U179), J (M304), L (M185), N (M231), O (M175), Q (M1105), R (P224) and T (M272). These genetic data allow one to predict the phenotype of the desired person according to the characteristics of eye, hair, skin color, AB0 blood group, sex, and genogeographic origin in the male line. The setting protocol is simplified as much as possible to facilitate the introduction of the method into practice. The distribution of allele frequencies of the studied polymorphisms, as well as AB0 blood groups among the Slavs (N = 482), originating mainly from central Russia, was established.

[Сomparison of immunoarrays for syphilis diagnostics produced by co-polymerization immobilization and non-contact printing techniques.]

The aim of the study was to investigate the characteristics of immunoarrays (microarrays) produced by co-polymerization immobilization and non-contact printing techniques for enhancing the capacities of syphilis diagnostics. In diagnostic context immunoarrays of both protein immobilization techniques have shown high sensitivity and specificity together with potency to differentiate syphilis stages in serologic assays. The article discloses the advantages and limitations of non-contact printing techniques as well as the results and problems revealed in the study. Solution of these problems in future may provide the development of new serodiagnostic tools with higher accuracy of the results.

[Diagnostic imminoarray assay for characterization of immunoglobulin igg and igm level in syphilis patients serum towards 12 recombinant antigens of t. pallidum before and after the therapy.]

The aim of the study was to characterize the dynamics of immunoglobulin IgG and IgM level in syphilis patients serum at different stages of the disease before and after the therapy towards 12 diagnostic antigens of T. pallidum in an microarray assay and to evaluate these data as possible prognostic markers. The dynamics of immunoglobulin IgG and IgM level was measured in the reaction of indirect immunofluorescence using microarray and compared to the results of non-treponemal RPR test and treponemal tests as EIA and reaction of passive hemagglutination. In microarray assay diagnostically high level of IgM in patients with primary, secondary and early latent and late latent syphilis decreased dramatically to zero after the successful therapy. Continuously high level of IgM after the therapy proposes the persistence of infection agents in the organism and points out the need of additional antimicrobial treatment. In most of the cases anti-treponemal IgG level also declined after the successful therapy and this confirms the appropriate treatment. The results of microarray assay coincide with the results of other mentioned laboratory tests for syphilis diagnostics. Microarray assay with the recombinant T. pallidum antigens gives the perspective for creating methods with wider spectrum of diagnostic and therapy control options using the IgM immunoglobulin level as a marker for successful syphilis treatment.

Immunochip for Syphilis Serodiagnostics with the Use of Extended Array of Treponema pallidum Recombinant Antigens.

An immunochip for multiple parallel detection of specific serum IgG in serological screening for syphilis is based on the use of an extended array of Treponema pallidum recombinant proteins and includes traditionally used immunodominant antigens (Tp15, Tp17, Tp47, and TmpA) and new synthetic proteins (Tp0277, Tp0319, Tp0453, Tp0684, Tp0965, and Tp1038). The use of individual antigens has demonstrated high analytical value of Tp0277 (periplasmatic C-terminal protease), Tp0319 (cytoplasmic membrane-associated lipoprotein TmpC), and external membrane-associated protein Tp0453 with transporting function, all of them improving significantly the efficiency of screening for syphilis in comparison with the traditional array of antigens. Multiparametric analysis of the results obtained on the immunochip with the use of linear discriminant analysis confirmed the efficiency of extended array of T. pallidum diagnostic antigens. Due to proposed modification, the "positive" and "negative" sera are clearly differentiated: the serological study showed 94.1% sensitivity and 100% specificity.

[Modification of Anti-Glycan IgG and IgM Profiles in Allergic Inflammation].

Glycans and anti-glycan antibodies (AGAs) are essential for infiltration of inflammatory cells in various allergies. The glycocalyx structure of the cells is modified during disease progression, and this modification is possible to evaluate by assessment of AGAs. A printed glycan array with 55 immobilized glycans and immobilized antibodies to IgG, IgA, and IgM was used to study the changes in AGA profiles in bronchial asthma (BA). Levels of antibodies to certain glycans in BA patients statistically differed from levels in healthy donors (p < 0.0007 by the Mann-Whitney test); the glycan set included 6Su-6`-SiaLec, Sia LeX, Sia6Htype2; Tαα, Manß1-4GlcNAc, and Manα1-4Manß. The obtained results help to better understand the mechanisms of the cell-mediated immune response in bronchial asthma and other types of allergic reactions.

Development of hydrogel biochip for in vitro allergy diagnostics.

A hydrogel biochip was developed for the simultaneous quantitative determination of sIgE for 21 allergens and total IgE in human serum. The biochips are manufactured by photoinduced copolymerization of different molecules (allergens and antibodies) with gel-forming monomers resulting in the formation of three-dimensional hydrogel elements (1nl gel drops). After incubation of the biochip with the serum, the results are visualized using fluorescently labeled anti-IgE antibodies. Using biochips, serum samples from allergic patients and healthy donors were analyzed and good correlation with the results obtained using commercial EIA test systems of generally recognized quality (Dr. Fooke Laboratorien GmbH, Germany) was observed.

[Sequence-specificity of protein-oligonucleotide interactions and development for protein isolation using sorptive medium with immobilized protein-specific oligonucleotides].

Sequence-specificity of binding of ribonuclease binase to oligodeoxyribonucleotides immobilized in biochip gel pads was studied. Binding constants for the complexes between binase and selected oligonucleotides were measured. Oligodeoxyribonucleotides GAGAGAG and GAGAGAGAG were found to be high-specific in interaction with binase. These oligonucleotides were used as molecular probes for immobilization in a sorptive medium for subsequent isolation and concentrating of binase from diluted water solutions. Volume capacity of the developed sorptive mediums containing immobilized oligodeoxyribonucleotides GAGAGAG and GAGAGAGAG was found to be 2.6 and 2.3 mg of binase per 1 mL of sorbate correspondingly. After the procedure of affinity chromatography and elution ofbinase from sorptive medium the protein showed the same affinity of binding to oligonucleotides as the initial sample.

Simultaneous detection of seven staphylococcal enterotoxins: development of hydrogel biochips for analytical and practical application.

A method of simultaneous analysis of staphylococcal enterotoxins using hydrogel-based microarrays (biochips) has been developed. The method allows simultaneous quantitative detection of seven enterotoxins: A, B, C1, D, E, G, and I in a single sample. The development of the method included expression and purification of recombinant toxins, production of panels of monoclonal antibodies (mAbs) against the toxins, and design and manufacturing of an experimental biochip for the screening of mAbs and selection of optimal pairs of primary and secondary antibodies for each toxin. The selected mAbs have high affinity toward their targets and no cross-reactivity with unrelated enterotoxins. Finally, a diagnostic biochip was designed for quantitative analysis of the toxins, and the analytical protocols were optimized. The sensitivity of the detection reached 0.1-0.5 ng/mL, depending on the type of enterotoxin. The evaluation of the resulting biochip using spiked food samples demonstrated that the sensitivity, specificity, and reproducibility of the proposed test system fully satisfy the requirements for traditional immunoanalytical systems. The diagnostic biochips manufactured on reflecting metal-coated surfaces shortened the time of analysis from 17 to 2 h without loss of sensitivity. The method was successfully tested on samples of food and biological media.

[Production and characteristics of monoclonal antibodies to the diphtheria toxin].

Monoclonal antibodies to the diphtheria toxin were produced without cross reactivity with the thermolabile toxin (LT) from Escherichia coli; ricin; choleraic toxin; the SeA, SeB, SeE, SeI, and SeG toxins of staphylococcus; the lethal factor of the anthrax toxin; and the protective antigen of the anthrax toxin. A pair of antibodies for the quantitative determination of the diphtheria toxin in the sandwich variation of enzyme-linked immunosorbent assay (ELISA) was chosen. The determination limit of the toxin was 0.7 ng/ml in plate and 1.6 ng/ml in microchip ELISA. The presence of a secretion from the nasopharynx lavage did not decrease the sensitivity of the toxin determination by sandwich ELISA. The immunization of mice with the diphtheria toxin and with a conjugate of the diphtheria toxin with polystyrene microspheres demonstrated that the conjugate immunization resulted in the formation of hybridoma clones which produced antibodies only to the epitopes of the A fragment of the diphtheria toxin. The immunization with the native toxin caused the production of hybridoma clones which predominantly produced antibodies to the epitopes of the B fragment.

[Preparation and characterization of monoclonal antibodies to the cholera toxin].

Monoclonal antibodies to cholera toxin were obtained. They do not cross-react with the termolabile toxin (LT) of Escherichia coli, ricin, diphtherial toxin, staphylococcus enterotoxins of SEA, SEB, SEI, SEG, or the lethal factor and protective antigen of the anthrax toxin. Pairs of antibodies for the quantitative measurement of the cholera toxin in sandwich enzyme immunoassay (EIA) were selected. The detection limit of the toxin is 0.2 ng/ml for plate EIA and 0.44 ng/ml for microchip EIA. The presence of milk, broth, or surface water in the toxin samples does not reduce the sensitivity of EIA.

[Development of a biochip for quantitative determination of two forms of prostate specific antigen using internal calibration curve].

Three-dimensional gel-based microchip allowing simultaneous quantitative detection of total (PSAtot) and free (PSAfree) forms of prostate specific antigen in human serum (in a format "one patient-one biochip") was developed. A method, which doesn't require preliminary construction of calibration curves when performing an assay, was applied for quantitative determination of PSAtot and PSAfree. Gel elements with immobilized antigen (PSA) in different concentration, forming an internal calibration curve, were included in a structure of the microchip, in addition to the elements with immobilized antibodies specific against PSAtot and PSAfree. The specialized software "ImaGelAssay" was used for data processing and interpretation. The sensitivity of the assay performed on biochips was 0.3 ng/ml for PSAtot and 0.2 ng/ml for PSAfree. Variation coefficient for the measurements inside one series of microchips didn't exceed 10%. Correlation coefficient between the results of measurements in human sera obtained on biochips and by the standard ELISA method was 0.988 for PSAtot and 0.987 for PSAfree.

[Evidence of species specific pericentric inversion in the karyotype of the midge Chironomus balatonicus]. / Dokazatel'stvo vidospetsificheskoi peritsentricheskoi inversii v kariotipe khironomidy Chironomus balatonicus.

Pericentric inversions do not play any important role in chromosomal rearrangements in the karyotype evolution of the genus Chironomus. However, a unique case of the fixed pericentric inversion was discovered in chromosome 2 of Chironomus balatonicus--one of the members of plumosus-species group (Kikhadze et al., 1996a; Golygina et al., 1996). According to morphological criteria, a centromere band on chromosome 2 changed its position in Ch. balatonicus. The cloned H3-SauDNA, specific for centromeres in plumosus group, was in situ hybridized with Ch. balatonicus polytene chromosomes, and thus a real change in the centromere position was proved to be a result of pericentric inversion. This was also confirmed after differential C-staining.

A molecular and cytogenetic analysis of lambda 20p7 fragment DNA from the proximal beta-heterochromatin of Drosophila melanogaster.

A DNA fragment from the Drosophila melanogaster genome, cloned in lambda 20p7, was derived independently from clones lambda 20 and lambda L [Baiborodin et al., Genetika 29 (1993) 403-416; Sharakhov et al., Genetika 29 (1993) 392-402]. In situ hybridization of lambda 20p7 DNA to the chromosomes of D. melanogaster demonstrated preferential hybridization of the fragment to the chromocenter of polytene chromosomes and to pericentric heterochromatin of chromosomes II, IV and X at the metaphase plate. Copy number per haploid genome for lambda 20p7 was estimated as approximately 200. Based on Southern blotting, the major portion of this moderate repeat was localized in the region of a 5.5-kb HindIII digest. In situ hybridization to polytene chromosomes from strain fs(2)B trophocytes revealed that repeats homologous to lambda 20p7 are located in the proximal heterochromatin which undergoes structural reorganization during tissue differentiation. The nucleotide sequence of two segments of the clone lambda 20p7, Dm0.9 and Dm270, was determined. Sequence analysis of the 300-bp Dm0.9 clone revealed that it contains 21-bp and 30-bp d(GT/CA) sequences, a 12-bp AT box, recognition sites for nuclear factors NFI and SpI, and a set of inverted repeats. Clone Dm270 contains an open reading frame (ORF). The deduced amino acid (aa) sequence shares homology with the gag-like gene from type-I (R1) ribosomal DNA insertion and may code for a polypeptide of 10 kDa. The Dm270 sequence was found to contain two direct repeats showing homology to the human CENP-B box.

The Chironomus thummi genome contains a non-LTR retrotransposon.

Nineteen recombinant phages containing DNA from the region of Balbiani ring a (BRa), which develops on chromosome IV in cells of the special lobe of the Chironomus thummi salivary gland, were isolated from a Chironomus thummi genomic library. Three of the clones contained transposable element sequences that hybridized to more than 100 sites on all four Chironomus chromosomes, including constant and variable sites. Two handogous clones, lambda 24 (which lacks the transposable element) and lambda 43 (which contains this insertion) were investigated by nucleotide sequence analysis. The complete nucleotide sequence of the 4.8 kb transposable element from Chironomus thummi (NLR1Cth) is reported here. This element contains two overlapping open reading frames of 1887 (ORF1) and 2649 bp (ORF2). Three cysteine motifs are found in the sequence of ORF1. Sequence similarity was found between ORF2 and known genes of viruses and transposable elements which encode reverse transcriptase. The NLR1Cth element has no long terminal repeats and is flanked by short direct repeats of the sequence TATCACTGACAAC. A 24 bp poly(dA) sequence was found at the 3' end of the element. Based upon its structural organization and comparative analysis of its nucleotide sequence we suggest that this NLR1Cth element belongs to the class of non-LTR retrotransposons. The genomic clone pC6.10 was previously obtained by microdissection and cloning of DNA from polytene chromosome IV of Chironomus thummi. A 2.4 kb insertion contained part of the 3' terminal region of the NLR1Cth element, but this differed in sequence from the first copy by several nucleotide substitutions and a shorter poly (dA) tract at the 3' end.(ABSTRACT TRUNCATED AT 250 WORDS)

[Drosophila beta-heterochromatin: molecular organization and function. Characteristics of the DNA sequences from proximal beta-heterochromatin, associated with the nuclear envelope of Drosophila melanogaster]. / beta-Geterochromatin Drosophila: molekuliarnaia organizatsiia i funktsionirovanie. Kharakteristika posledovatel'nosti DNK iz proksimal'nogo beta-geterokhromatina, assotsiirovannogo s iadernoi obolochkoi Drosophila melanogaster.

To study the nucleotide sequence from the pericentric heterochromatin associated with the nuclear envelope, a residual DNA was extracted from the DNAse-treated nuclear lamins of Drosophila melanogaster tissue culture cell line Kc. The isolated DNA was cloned in lambda vector. The DNA library obtained was screened for the clones homologous to the pericentric heterochromatin. The experiments on in situ hybridization to the polytene chromosome of the nurse cell nuclei of the strain fs(2) B assigned the reiterated sequence, homologous to the lamin DNA clone, to the nuclear envelope associated regions of the proximal beta-heterochromatin which is known to undergo structural reorganization during cell differentiation. The nucleotide analysis of 300 bp from this sequence has established the presence of 21 bp and 300 bp d(GT/CA), 12 bp AT-box, the regions of recognition of the nuclear factor and the inverted repeats.