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The family of genes containing C2H2 zinc finger domains, which has more than 700 members, is one of the largest in the genome. Of particular interest are C2H2 genes with potential tissue-specific transcription, which determine the functional properties of individual cell types, including those associated with pathological processes. The aim of this work was to identify C2H2 family genes with tissue-specific transcription and analyze changes in their activity during tumor progression. To search for these genes, we used four databases containing data on gene transcription in human tissues obtained by RNA-Seq analysis. The analysis showed that, although the major part of the C2H2 family genes is transcribed in virtually all tissues, a group of genes has tissue-specific transcription, with most of the transcripts being found in the testis. After having compared all four databases, we identified nine such genes. The testis-specific transcription was confirmed for two of them, namely ZBTB32 and ZNF473, using quantitative PCR of cDNA samples from different organs. A decrease in ZBTB32 and ZNF473 transcription levels was demonstrated in germ cell tumors. The studied genes can serve as candidate markers in germ cell tumors.

One of the most popular nonviral delivery systems for gene therapy constructs are carriers based on polyethylenimine (PEI) DNA complexes. A number of disadvantages associated with the lack of targeted delivery and increased cytotoxicity are overcome by adding auxiliary molecules to the complexes. An example of this is chondroitin sulfate (CS). The purpose of this work was to assess the effect of CS on the transfection properties of DNA–PEI complexes under different conditions of their preparation and transfection protocols. All complexes were prepared in solutions with high and low ionic strength. Transfection of C26 cells was performed according to two protocols differing in the presence of serum in the medium. The portion of transfected cells, transgene expression level, and cell viability were the main parameters of assessing the transfection efficiency. In binary DNA–PEI complexes prepared in different salt conditions, using different transfection protocols, the difference in the portion of transfected cells reached ten times. Addition of CS improved this transfection efficiency indicator up to 6.5 times, while the maximum difference in this indicator for the corresponding ternary complexes was reduced to 2.5 times. Changes in the proportion of CS in the composition of the complexes had an insignificant effect on their transfection properties. In the case of complexes prepared in high ionic strength solutions, the order of CS addition was also important. The best results of transfection efficiency were achieved with ternary complexes prepared in low ionic strength solutions, using a serum-free protocol, while these indicators were comparable with the data for Lipofectamine 2000. The addition of chondroitin sulfate improves the transfection properties of DNA–PEI complexes and makes them less dependent on the methods of preparation and transfection.

The embryonic development of all body systems is controlled by coordinated interactions of a group of genes encoding master regulators of development, which determine the fate of cells in the process of differentiation. These genes are often involved in the processes of malignant degeneration of cells. Hence, they are considered as targets of targeted gene therapy in the development of modern approaches to cancer treatment. One of the most aggressive forms of cancer is pancreatic ductal adenocarcinoma (PDAC). Thus, the study of pancreas master regulators genes is a challenging task. This review is devoted to SOX9, which is one of the key master regulators of pancreatic development and is a crucial factor in the induction and progression of PDAC.

The emergence of genetic diseases and evolutionary processes are associated with the flow of genetic information from one generation to another, in which genetic information carried by gametes can be changed by the appearance of de novo germline mutations. The rate of germline mutations determines the rate of evolution and the incidence of heritable disorders. Despite the great theoretical and practical importance, the problem of establishing mutation rates and their dependence on different factors remains scarcely studied, and the mutation rate values obtained by different methods vary considerably. The review discusses different ways of estimating the rate of these mutations and makes an attempt to explain the reasons for discrepancies in the data obtained. Three levels of the mutation formation are considered: (1) mutations that are formed during the development of a given individual during gametogenesis (basic mutations); (2) mutations transmitted to offspring and determining differences in the genomes of consecutive generations (parents and offspring), which include basic mutations and possible changes resulting from complex processes of sperm transfer to oocyte, fertilization, and subsequent events that lead to only one viable offspring of hundreds of millions spermatozoa and oocytes; (3) mutations which are formed at level 2, fixed in evolution, and determine evolutionary processes and differences between genomes, in particular, of hominoids, hominids, and hominins.

Earlier, it was reported that the strong cytomegalovirus enhancer can activate the cytomegalovirus promoter in trans, i.e. as a separate plasmid co-transfected with a promoter-reporter gene construct. Here we demonstrate that the ability of enhancers to activate promoters in trans in transient transfection experiments is a property of not only viral regulatory elements but also of various genomic enhancers and promoters. Enhancer-promoter activation in trans is promoter- and cell type-specific, and accompanied by physical interaction between promoter and enhancer as revealed by chromosome conformation capture assays. Thus, promoter activation in transient co-transfection of promoters and enhancers shares a number of important traits with long-distance promoter activation by enhancers in living cells and may therefore serve as a model of this fundamental cellular process.

A systematic search for DNA fragments containing potential CTCF transcription factor binding sites in the chicken alpha-globin domain and its flanking regions was performed by means of the two-dimension electrophoretic mobility shift assay. For the alpha-globin domain fragments selected, the occupancy by the CTCF in erythroid and lymphoid chicken cells was tested by chromatin immunoprecipitation. Only one of 13 DNA fragments capable of CTCF binding in vitro was efficiently bound to this protein in vivo in erythroid cells, and somewhat less efficiently - in lymphoid cells. So, binding of CTCF to the DNA fragment in vitro in most cases does not mean that this fragment will be occupied by CTCF in the cell nucleus. Yet, CTCF binding in vivo, as a rule, is accompanied by the binding of the protein to this DNA region in vitro. During the erythroid differentiation, no significant changes in CTCF binding to the DNA fragments studied were detected.

The CTCF transcription factor is thought to be one of the main participants in various gene regulatory networks including transcription activation and repression, formation of independently functioning chromatin domains, regulation of imprinting etc. Sequencing of human and other genomes opened up a possibility to ascertain the genomic distribution of CTCF binding sites and to identify CTCF-dependent cis-regulatory elements, including insulators. In the review, we summarized recent data on CTCF functioning within a framework of the chromatin loop domain hypothesis of large-scale regulation of the genome activity. Its fundamental properties allow CTCF to serve as a transcription factor, an insulator protein and a dispersed genome-wide demarcation tool able to recruit various factors that emerge in response to diverse external and internal signals, and thus to exert its signal-specific function(s). Фактор транскрипції CTCF вважають одним з основних учасни- ків різних мереж регуляції генів, з-поміж яких активація і репресія транскрипції, утворення незалежно функціонуючих доменів хроматину, регуляція імпринтингу тощо. Секвенування геномів людини та інших организмів дозволяє виявляти геномний розподіл сайтів зв’язування CTCF та ідентифікувати CTCF-залежні регуляторні елементи, до яких належать інсулятори. В огляді підсумовано нові дані з функціонування CTCF у рамках гіпотези участі петельних доменів хроматину у великомасштабній регуляції активності геному. Фундаментальні властивості CTCF дозволяють йому діяти як регулятор транскрипції, інсуляторний білок, а також як розподілений по геному прикордонний елемент,здатний залучати різні фактори, які з’являються у відповідь на різноманітні зовнішні та внутрішні чинники і таким чином виконувати свої сигнал-специфічні функції. Фактор транскрипции CTCF считается одним из основных участников различных сетей регуляции генов, включая активацию и репрессию транскрипции, образование независимо функционирующих доменов хроматина, регуляцию импринтинга и т. д. Секвенирование геномов человека и других организмов позволяет выявить геномное рапределение сайтов связывания CTCF и идентифицировать CTCF-зависимые регуляторные элементы, включая инсуляторы. В обзоре суммированы новые данные по функционированию CTCF в рамках гипотезы участия петельных доменов хроматина в крупномасштабной регуляции активности генома. Фундаментальные свойства CTCF позволяют ему действовать как регулятор транскрипции, инсуляторный белок, а также как распределенный по геному пограничный элемент, способный привлекать различные факторы, появляющиеся в ответ на разнообразные внешние и внутренние воздействия и таким образом осуществлять свои сигнал-специфичные функции.

The chicken gene for transcription factor CTCF was expressed in COS-1 mammalian cells. The CTCF protein containing polyhistidine tag was partially purified using metallo-affinity and ion-exchange chromatography. The expressed protein localized in the cell nucleus and was shown to be functionally active in the electrophoretic mobility shift assay and specifically interacted with anti-CTCF antibodies.

We used a transient transfection of cultured cells with linearized plasmids to analyze the enhancer-blocking activity of potential insulators including the standard cHS4 chicken beta-globin insulator and several DNA fragments selected from the human genome sequence. About 60-80% of the potential insulators do reveal the enhancer-blocking activity when probed by the transient transfection assay. The activity of different sequences is characterized by certain tissue specificity and by dependence on the orientation of the fragments relative to the promoter. Thus, the transfection model may be used for quantitative analysis of the enhancer-blocking activity of the potential insulators.

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 in mixtures.

The CTCF transcription factor is an 11 zinc fingers multifunctional protein that uses different zinc finger combinations to recognize and bind different sites within DNA. CTCF is thought to participate in various gene regulatory networks including transcription activation and repression, formation of independently functioning chromatin domains and regulation of imprinting. Sequencing of human and other genomes opened up a possibility to ascertain the genomic distribution of CTCF binding sites and to identify CTCF-dependent cis-regulatory elements, including insulators. In the review, we summarized recent data on genomic distribution of CTCF binding sites in the human and other genomes within a framework of the loop domain hypothesis of large-scale regulation of the genome activity. We also tried to formulate possible lines of studies on a variety of CTCF functions which probably depend on its ability to specifically bind DNA, interact with other proteins and form di- and multimers. These three fundamental properties allow CTCF to serve as a transcription factor, an insulator and a constitutive dispersed genome-wide demarcation tool able to recruit various factors that emerge in response to diverse external and internal signals, and thus to exert its signal-specific function(s).

In this study, we have identified 20 human sequences containing Myc network binding sites in a library from the whole human chromosome 19. We demonstrated binding of the Max protein to these sequences both in vitro and in vivo. The majority of the identified sequences contained one or several CACGTG or CATGTG E-boxes. Several of these sites were located within introns or in their vicinity and the corresponding genes were found to be up- or down-regulated in differentiating HL-60 cells. Our data show the proof of principle for using this strategy in identification of Max target genes, and this method can also be applied for other transcription factors.

We report a new experimental method of direct selection, identification, and mapping of potential enhancer sequences within extended stretches of genomic DNA. The method allows simultaneous cloning of a quantity of sequences instead of tedious screening of the separate ones, thus providing a robust and high-throughput approach to the mapping of enhancers. The selection procedure is based on the ability of such sequences to activate a minimal promoter that drives expression of a selective gene. To this end a mixture of short DNA fragments derived from the segment of interest was cloned in a retroviral vector containing the neomycin phosphotransferase II gene under control of a cytomegalovirus (CMV) minimal promoter. The pool of retroviruses obtained was used to infect HeLa cells and then to select neomycin-resistant colonies containing constructs with enhancer-like sequences. The pool of the genomic fragments was rescued by PCR and cloned, forming a library of the potential enhancers. Fifteen enhancer-like fragments were selected from 1-Mb human genome locus, and enhancer activity of 13 of them was verified in a transient transfection reporter gene assay. The sequences selected were found to be predominantly located near 5′ regions of genes or within gene introns.

Genome sequencing in human and other organisms make it possible to map and analyze the regulatory elements of the genome. Yet there are only scarce experimental data on mapping regulatory elements, such as enhancers, silencers, insulators, transcription terminators, and replication origins, especially at the level of the whole genome. In silico prediction of the positions of such elements is rather difficult because their functions are often tissue-or cell-specific. Hence, it is important to develop high-throughput experimental approaches to the identification and mapping of the functional elements of the genome. The review considers such approaches developed for mammalian genomes, as the identification of their regulatory elements is essential for complete functional genome annotation.

The availability of complete human and other metazoan genome sequences has greatly facilitated positioning and analysis of various genomic functional elements, with initial emphasis on coding sequences. However, complete func- tional maps of sequenced eukaryotic genomes should include also positions of all non-coding regulatory elements. Unfor- tunately, experimental data on genomic positions of a multitude of regulatory sequences, such as enhancers, silencers, in- sulators, transcription terminators, and replication origins are very limited, especially at the whole genome level. Since most genomic regulatory elements (e.g. enhancers) are generally gene-, tissue-, or cell-specific, the prediction of these elements by computational methods is difficult and often ambiguous. Therefore, the development of high-throughput ex- perimental approaches for identifying and mapping genomic functional elements is highly desirable. At the same time, the creation of whole-genome map of hundreds of thousands of regulatory elements in several hundreds of tissue/cell types is presently far beyond our capabilities. A possible alternative for the whole genome approach is to concentrate efforts on individual genomic segments and then to integrate the data obtained into a whole genome functional map. Moreover, the maps of polygenic fragments with functional cis-regulatory elements would provide valuable data on complex regulatory systems, including their variability and evolution. Here, we reviewed experimental approaches to the realization of these ideas, including our own developments of experimental techniques for selection of cis-acting functionally active DNA fragments from large (megabase-sized) segments of mammalian genomes.

There is abundant evidence that the DNA in eukaryotic cells is organized into loop domains that represent basic structural and functional units of chromatin packaging. To explore the DNA domain organization of the breast cancer loss-of-heterozygosity region on human chromosome 16q22.1, we have identified a significant portion of the scaffold/matrix attachment regions (S/MARs) within this region. Forty independent putative S/MAR elements were assigned within the 16q22.1 locus. More than 90% of these S/MARs are AT rich, with GC contents as low as 27% in 2 cases. Thirty-nine (98%) of the S/MARs are located within genes and 36 (90%) in gene introns, of which 15 are in first introns of different genes. The clear tendency of S/MARs from this region to be located within the introns suggests their regulatory role. The S/MAR resource constructed may contribute to an understanding of how the genes in the region are regulated and of how the structural architecture and functional organization of the DNA are related.

Specific binding of nuclear proteins, in particular transcription factors, to target DNA sequences is a major mechanism of genome functioning and gene expression regulation in eukaryotes. Therefore, identification and mapping specific protein target sites (PTS) is necessary for understanding genomic regulation. Here we used a novel two-dimensional electrophoretic mobility shift assay (2D-EMSA) procedure for identification and mapping of 52 PTS within a 563-kb human genome region located between the FXYD5 and TZFP genes. The PTS occurred with approximately equal frequency within unique and repetitive genomic regions. PTS belonging to unique sequences tended to group together within gene introns and close to their 5′ and 3′ ends, whereas PTS located within repeats were evenly distributed between transcribed and intragenic regions.

The spatial organization of an approximately 170 kb region of human chromosome 19, including CD22 and GPR40-GPR43 genes, was studied using in situ hybridization of a set of cosmid and PAC probes with nuclear halos prepared from proliferating and differentiated HL60 cells. The whole region under study was found to be looped out into the nuclear halo in proliferating cells. It is likely that the loop observed was attached to the nuclear matrix via MAR elements present at the flanks of the area under study. Upon dimethyl sulfoxide-induced differentiation of the cells the looped fragment became associated with the nuclear matrix. This change in the spatial organization correlated with the activation of transcription of at least two (CD22 and GPR43) genes present within the loop. The data obtained are discussed in the framework of the hypothesis postulating that the spatial organization of chromosomal DNA is maintained via constitutive (basic) and facultative (transcription-related) interactions of the latter with the nuclear matrix.

Transient expression of a luciferase reporter gene was used to evaluate tissue-specific promoter and enhancer activities of a solitary extraviral long terminal repeat (LTR) of the human endogenous retrovirus K (HERV-K) in several human and CHO cell lines. The promoter activity of the LTR varied from virtually not detectable (GS and Jurkat cells) to as high as that of the SV40 early promoter (Tera-1 human testicular embryonal carcinoma cells). The negative regulatory element (NRE) of the LTR retained its activity in all cell lines where the LTR could act as a promoter, and was also capable of binding host cell nuclear proteins. The enhancer activity of the LTR towards the SV40 early promoter was detected only in Tera-1 cells and was not observed in a closely related human testicular embryonal carcinoma cell line of different origin, NT2/D1. A comparison of proteins bound to central part of the LTR in nuclear extracts from Tera-1 and NT2/D1 by electrophoretic mobility shift assay revealed striking differences that could be determined by different LTR enhancer activities in these cells. Tissue specificity of the SV40 early promoter activity was also revealed.

The transient expression of the luciferase reporter gene was used to detect the tissue-specific enhancer activity of the solitary extraviral long terminal repeat (LTR) of the human endogenous retrovirus K (HERV-K). The LTR was previously mapped to the 19q13.2 locus. It contains a number of potential regulatory elements including TATA box, binding sites for some nuclear factors, and a polyadenylation signal. However, an analysis of the genomic sequences close to the LTR did not reveal any known genes or the expressed sequences (EST), whose functioning could be regulated by this LTR. The enhancer activity can be preserved in the solitary LTR due to its involvement in the long-range control of genome functioning or by the absence of functional disruptive mutations within the human-specific LTR, because it is of a relatively young evolutionary age.

The first draft human genome sequence now available allowed the identification of an enormous number of gene coding areas of the genomic DNA. However, a great number of regulatory elements such as enhancers, promoters, transcription terminators, or replication origins can not be identified unequivocally by their nucleotide sequences in complex eukaryotic genomes. One important subclass of these type of sequences is scaffold/matrix attachment regions (S/MARs) that were hypothesized to anchor chromatin loops or domains to the nuclear matrix and/or chromosome scaffold. We developed an experimental selection procedure to identify S/MARs within a completely sequenced one megabase (1 Mb) long gene-rich D19S208-COX7A1 locus of human chromosome 19. A library of S/MAR elements from the locus was prepared and shown to contain -20 independent S/MARs. Sixteen of them were isolated, sequenced, and assigned to certain positions within the locus. A majority of the S/MARs identified (11 out of 16) lie in intergenic regions, suggesting their structural role, i.e., delimitation of chromatin domains. These 11 S/MARs subdivide the locus into 10 domains ranging from 6 to 272 kb with an average domain size of 88 kb. The remaining five S/MARs were found within intronic sequences of APLP1, HSPOX1, MAG, and NPHS1 genes, and can be tentatively characterized as regulatory S/MARs. The correspondence of the chromatin domains defined by the S/MARs to functional characteristics of the genes therein is discussed. The approach described can be a prototype of a similar search of long sequenced genomic stretches and/or whole chromosomes for various regulatory elements.

We used the 4C-Seq technique to characterize the genome-wide patterns of spatial contacts of several CpG islands located on chromosome 14 in cultured chicken lymphoid and erythroid cells. We observed a clear tendency for the spatial clustering of CpG islands present on the same and different chromosomes, regardless of the presence or absence of promoters within these CpG islands. Accordingly, we observed preferential spatial contacts between Sp1 binding motifs and other GC-rich genomic elements, including the DNA sequence motifs capable of forming G-quadruplexes. However, an anchor placed in a gene/CpG island-poor area formed spatial contacts with other gene/CpG island-poor areas on chromosome 14 and other chromosomes. These results corroborate the two-compartment model of the spatial organization of interphase chromosomes and suggest that the clustering of CpG islands constitutes an important determinant of the 3D organization of the eukaryotic genome in the cell nucleus. Using the ChIP-Seq technique, we mapped the genome-wide CTCF deposition sites in the chicken lymphoid and erythroid cells that were used for the 4C analysis. We observed a good correlation between the density of CTCF deposition sites and the level of 4C signals for the anchors located in CpG islands but not for an anchor located in a gene desert. It is thus possible that CTCF contributes to the clustering of CpG islands observed in our experiments.

Solitary long terminal repeats (LTRs) of human endogenous retroviruses (HERVs), tens of thousands of which are spread all over the genome, contain a variety of potential transcription regulatory elements. Information on transcriptional behavior of individual solitary LTRs, however, is limited. We studied the transcriptional activity of several individual HERV-K LTRs in a variety of tissues and cell lines. The RT-PCR technique targeted at specific amplification of the U3 or U5 regions of individual LTRs together with their unique genomic flanks was used to estimate the content of each region in the transcripts. An unequal abundance of the U3 and U5 regions in the transcripts of the same LTR in different cells and tumors was observed. Each LTR is transcribed differently in different cells or tissues, and transcriptional behavior of different LTRs was different in the same cell line or tissue. The transcriptional status of LTRs varies in response to mitogenic and stress factors and in tumor tissues compared to normal counterparts. The LTRs thus seem to be the subjects of specific transcription regulation. The data obtained indicate that an appreciable fraction of the LTRs retained regulatory potential throughout millions of years of evolution and thus may contribute to the overall transcription regulatory network.

Intrinsic lysozyme-like activity was demonstrated for destabilase from the medicinal leech supported by (1) high specific lysozyme activity of the highly purified destabilase, (2) specific inhibition of the lysozyme-like activity by anti-destabilase antibodies, and (3) appreciable lysozyme-like activity in insect cells infected with recombinant baculoviruses carrying cDNAs encoding different isoforms of destabilase. Several isoforms of destabilase constitute a protein family at least two members of which are characterized by lysozyme activity. The corresponding gene family implies an ancient evolutionary history of the genes although the function(s) of various lysozymes in the leech remains unclear. Differences in primary structures of the destabilase family members and members of known lysozyme families allow one to assign the former to a new family of lysozymes. New proteins homologous to destabilase were recently described for Caenorhabditis elegans and bivalve mollusks suggesting that the new lysozyme family can be widely distributed among invertebrates. It remains to be investigated whether the two enzymatic activities (isopeptidase and lysozyme-like) are attributes of one and the same protein.

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.

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.

Using a novel procedure a representative human chromosome 19-specific library was constructed of short sequences, which bind preferentially to the nuclear matrix (matrix attachment regions, or MARs). Judging by 20 clones sequenced so far, the library contains > 50% of human inserts, about 90% of which are matrix-binding by the in vitro test. Computer analysis of sequences of eight human MARs did not reveal any significant homologies with the EMBL Nucleotide Data Base entries as well as between MARs themselves. Eight MARs were assigned to individual positions on the chromosome 19 physical map. The library constructed can serve as a good source of MAR sequences for comparative analysis and classification and for further chromosome mapping of MARs as well.

Head-to-head genes with a short distance between their transcription start sites may constitute up to 10% of all genes in the genomes of various species. It was hypothesized that this intergenic space may represent bidirectional promoters which are able to initiate transcription of both genes, but the true bidirectionality was proved only for a few of them. We present experimental evidence that, according to several criteria, a 269 bp region located between the PSENEN and U2AF1L4 human genes is a genuine bidirectional promoter regulating a concerted divergent transcription of these genes. Concerted transcription of PSENEN and U2AF1L4 can be necessary for regulation of T-cell activity.

A detailed functional and evolutionary analysis of an enhancer element of the human genome (enhancer 12) located in the second intron of the U2AF1L4 gene, which we identified earlier, is presented. Overlapping fragments of the studied genome region were analyzed for enhancer activity, and the site responsible for the activity of this element was identified using transient transfections of HeLa cells. Comparison of the enhancer 12 sequence with orthologous sequences from seven primate species revealed the existence of evolutionarily conserved sequences within this element. One of the identified conservative regions is likely responsible for the enhancer activity and is able to specifically interact in vitro with proteins of HeLa cell nuclear extract. The ability of orthologous primate sequences to compete with enhancer 12 for binding with HeLa cell nuclear extract proteins and to enhance the activity of the reporter gene in transient transfection of HeLa cells is demonstrated.

For a 140-kb human genome locus, an analysis of the distribution of Dam methylase accessible sites, DNase I sensitive and resistant regions, unmethylated CpG sites and acetylated histone H3 molecules was performed and compared with transcriptional activity of the genes within the locus. A direct correlation was found between the extent of Dam methylation and C5 cytosine (CpG) methylation. It was also demonstrated that promoter regions of all highly and moderately transcribed genes are highly accessible to methylation by Dam methylase. In contrast, promoters of non-transcribed genes showed a very low extent of Dam methylation. Promoter regions of non-transcribed genes were also highly CpG methylated, and the promoter and more distant 5'-regions of the housekeeping gene COX6B1 were substantially CpG-demethylated. Some highly Dam methylase accessible regions are present in the intergenic regions of the locus suggesting that the latter contain either unidentified non-coding transcripts or extended regulatory elements like locus control regions.

A positive-negative selection system revealed 10 potential insulators able to block enhancer interaction with promoter in the 10(6) bp human chromosome 19 region between genes FXYD5 and COX7A1. Relative positions of insulators and genes are in accord with the hypothesis that insulators subdivide genomic DNA into independently regulated loop domains.

The publication of the human and other metazoan genome sequences opened up the possibility for mapping and analysis of genomic regulatory elements. Unfortunately, experimental data on genomic positions of such sequences as enhancers, silencers, insulators, transcription terminators, and replication origins are very limited, especially at the whole genome level. As most genomic regulatory elements (e.g., enhancers) are generally gene-, tissue-, or cell-specific, the prediction of these elements in silico is often ambiguous. Therefore, the development of high-throughput experimental approaches for identification and mapping of genomic functional elements is highly desirable. In this review we discuss novel approaches to high-throughput experimental identification of mammalian genomes cis-regulatory elements which is a necessary step toward the complete genome annotation.

Epigenetic elements of the genome, i.e. elements that determine stably inherited changes in gene expression without changes in the genomic DNA sequence, are essential tools of genetic regulation in higher eukaryotes. The complete sequencing of the human and other genomes allowed studies to be started on positioning of these elements within long multigenic regions of the genome, which is a prerequisite for a comprehensive functional annotation of genomes. This mini-review considers some recent experimental approaches to the high-throughput identification and mapping of epigenetic elements of mammalian genomes, including the mapping of methylated CpG sites, open and closed chromatin regions, and DNase I hypersensitivity sites.

We developed a technique of differential electrophoretic mobility shift assay (EMSA) display allowing identification of tissue-specific protein-binding sites within long genomic sequences. Using this approach, we identified 10 cell type-specific protein-binding sites (protein target sites [PTSs]) within a 137-kb human chromosome 19 region. In general, tissue-specific binding of proteins from different nuclear extracts by individual PTSs did not follow the all-or-nothing principle. Most often, PTS-protein complexes were formed in all cases, but they were different for different nuclear extracts used.

In this study, we compared degree of methylation of selected CpG sites in CCGG sequences located in promoter regions of four human genes with expression level of these genes in several human cell lines and tissues. These genes were subdivided into two groups according to the dependence of their expression on CpG methylation in the 5′-regions. The first group, characterized by clear correlation of methylation with the transcription level, includes housekeeping gene COX6B (the absence of methylation unambiguously correlates with expression) and urothelium-specific uroplakin gene (the methylation coincides with absence of expression). The second group includes genes that are expressed in many, but not all tissues and cells. For these genes (LEAP-1 and ATP4A), there was no correlation between methylation and expression. It is possible that methylation provides some basal level of gene repression, which is overcome by binding of tissue-specific transcription factors, whereas lack of methylation gives the opportunity for gene expression in various cells and tissues.

Identification of insulators is one of the most difficult problems in functional mapping of genomes. For this reason, up to now only a few insulators have been described. In this article we suggest an approach that allows direct isolation of insulators by a simple positive-negative selection based on blocking enhancer effects by insulators. The approach allows selection of fragments capable of blocking enhancers from mixtures of genomic fragments prepared from up to 1-Mb genomic regions. Using this approach, a 1-Mb human genome locus was analyzed and eight potential insulators were selected. Five of the eight sequences were positioned in intergenic regions and two were within introns. The genes of the alpha-polypeptide H+/K+ exchanging ATPase (ATP4A) and amyloid beta (A4) precursor-like protein 1 (APLP1) within the locus studied were found to be flanked by insulators on both sides. Both genes are characterized by distinct tissue-specific expression that differs from the tissue specificity of the surrounding genes. The data obtained are consistent with the conception that insulators subdivide genomic DNA into loop domains that comprise genes characterized by similar expression profiles. Using chromatin immunoprecipitation assay, we demonstrated also that at least six of the putative insulators revealed in this work could bind the CTCF transcription factor in vivo. We believe that the proposed approach could be a useful instrument for functional analysis of genomes.

An approach for fast identification and mapping of transcription factor binding sites within long genomic sequences is proposed. Using this approach, 10 CCCTC-binding factor (CTCF) binding sites were identified within a 1-Mb FXYD5-COX7A1 human chromosome 19 region. In vivo binding of CTCF to these sites was verified by chromatin immunoprecipitation assay. CTCF binding sites were mapped within gene introns and intergenic regions, and some of them contained Alu-like repeated elements.

S/MARs (scaffold/matrix attachment regions) are the DNA regions that are involved in the interaction with the nuclear matrix and are identified by in vitro methods. According to the available information, S/MARs possess an insulating activity, i.e., the ability to block the interaction between the enhancer and promoter in vivo, and are, probably, intact insulators or their fragments. Nevertheless, there is still no direct proof for this correspondence. To obtain additional information on the insulator activity of S/MARs, we selected five DNA fragments of different lengths and affinities for the nuclear matrix from the previously constructed library of S/MARs and tested their ability to serve as insulators. Two of five elements exhibited an insulator (enhancer-blocking) activity upon the transient transfection of CHO cells. None of the S/MARs displayed either promoter or enhancer/silencer activities in these cells.

Destabilase-lysozyme (DL) from salivary gland secretion of the medicinal leech (Hirudo medicinalis) is as a member of the invertebrate lysozyme family, which sharply differs from other lysozyme families. In this study, DL lysozyme function was confirmed during expression of a gene encoding DL in Escherichia coli. Several constructs of the expression vectors pKK OmpA and pET-3A with or without bacterial, leech, or yeast signal peptides (SP) were used. The use of a construct without signal peptide genes resulted in normal growth of the transformed cells. Transformation of E. coli cells with the constructs containing SP was accompanied by the disruption of the forming cells. The use of the expression vector pET-32 LTC-System for production of DL as a fusion protein with thioredoxin also resulted in normal cell growth. However, specific activity of DL isolated from such cells was significantly lower than that of enzyme purified from extracts of Spodoptera frugiperda cells, which were infected with the baculovirus vector carrying DL cDNA. It is shown that the action mechanism of invertebrate lysozyme does not differ from that of other families: recombinant DL from S. frugiperda extracts catalyzed cleavage of synthetic substrate, hexamer of N-acetylglucosamine, to di- and tetramers, which is typical for enzymatic function of other lysozyme families.

Modern concepts on the chromatin loop-domain organization and the role of the DNA regions specifically binding the nuclear matrix (nuclear scaffold, or S/MARs) in its formation, maintenance, and regulation are discussed. Some S/MAR structural features, properties of binding the nuclear matrix, and probable mechanisms of their involvement in regulation of gene activity are considered. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 1; see also http://www.maik.ru.

The transient expression of the luciferase reporter gene helped us to detect a tissue-specific enhancer activity of the solitary extraviral long terminal repeat (LTR) of the human endogenous retrovirus K (HERV-K). The LTR was previously mapped to the 19q13.2 locus. It contains a number of potential regulatory elements including TATA box, binding sites for some nuclear factors, and a polyadenylation signal. However, an analysis of the genomic sequences close to the LTR did not reveal any known genes or the expressing marker sequences (EST), whose functioning could be regulated by this LTR. The enhancer activity can be preserved in the solitary LTR due to its involvement in a long-range control of genome functioning or by the absence of functional disruptive mutations within the human-specific LTR, because it is of a relatively young evolutionary age. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2002, vol. 28, no. 4; see also http://www.maik.ru.

The first draft human genome sequence now available allowed the identification of an enormous number of gene coding areas of the genomic DNA. However, a great number of regulatory elements such as enhancers, promoters, transcription terminators, or replication origins can not be identified unequivocally by their nucleotide sequences in complex eukaryotic genomes. One important subclass of these type of sequences is scaffold/matrix attachment regions (S/MARs) that were hypothesized to anchor chromatin loops or domains to the nuclear matrix and/or chromosome scaffold. We developed an experimental selection procedure to identify S/MARs within a completely sequenced one megabase (1 Mb) long gene-rich D19S208-COX7A1 locus of human chromosome 19. A library of S/MAR elements from the locus was prepared and shown to contain -20 independent S/MARs. Sixteen of them were isolated, sequenced, and assigned to certain positions within the locus. A majority of the S/MARs identified (11 out of 16) lie in intergenic regions, suggesting their structural role, i.e., delimitation of chromatin domains. These 11 S/MARs subdivide the locus into 10 domains ranging from 6 to 272 kb with an average domain size of 88 kb. The remaining five S/MARs were found within intronic sequences of APLP1, HSPOX1, MAG, and NPHS1 genes, and can be tentatively characterized as regulatory S/MARs. The correspondence of the chromatin domains defined by the S/MARs to functional characteristics of the genes therein is discussed. The approach described can be a prototype of a similar search of long sequenced genomic stretches and/or whole chromosomes for various regulatory elements.

From a library of sequences binding preferentially to nuclear matrix (matrix attachment regions, MARs), a fragment of about 300 bp in length (CEA (carcinoembryonic antigen)-MAR) was isolated and characterized. The CEA-MAR sequence was found in more than ten loci of chromosome 19 containing elements similar to genes of the CEA family. No sequences of this group were found on other human chromosomes. Two CEA-MAR-containing loci were sequenced, and sequences for another seven loci were found in GenBank. A comparative analysis of CEA-MARs and the flanking sequences is reported. Based on the sequence of the CEA-containing chromosome 19 loci, a hypothetical model of the domain structure of a 2-Mb chromosome region was constructed and the mutual arrangement of CEA-MARs and genes of CEA family was elucidated. The CEA-MARs were located 5-20 kb downstream of the CEA genes. These results suggest that the duplication unit of the CEA family may coincide with chromatin domains containing these genes.

Our previous studies showed a predominance of high molecular weight protein group in tumor nuclear matrices. Contrary to normal cells, proteins of this group are preferentially phosphorylated. Phosphoproteins of hepatoma nuclear matrix are selectively subjected to rapid proteolysis. By alkali treatment and a monoclonal antibody against phosphotyrosyl residue the presence of two high molecular weight bands of phosphotyrosyl-containing proteins was detected in nuclear matrices of tumor but not of normal liver cells. High molecular weight protein group of tumor nuclear matrices revealed also a rapid turnover and preferential incorporation of labeled amino acids selectively inhibited by chloramphenicol.

Solitary long terminal repeats (LTRs) of the human endogenous retroviruses, scattered in several thousand copies throughout the human genome, are potentially capable of affecting the expression of closely located genes. To assess their regulatory potential, the LTR sequences of one of the most abundant HERV families (HERV-K) were screened for the presence of binding sites for the host cell nuclear factors using mobility shift and UV-crosslinking assays. It was shown that the LTR sequences of two subfamilies harbor a specific binding site for a complex consisting of at least three proteins, ERF1, ERF2 and ERF3 of 98, 91 and 88 kDa apparent molecular mass, respectively. This binding site is located in the 5′ region of the LTR U3 element. The preservation of the specific protein binding site in different HERV-K LTR sequences suggests their possible role in regulation of nearby located genes.

A plasmid library (400 clones) of the human chromosome 19 sequences selectively binding to nuclear matrix was obtained and characterized. Approximately 50% clones contained DNA sequences specific for human chromosome 19. Over 90% of these sequences represented matrix attachment regions (MARs). Eight MARs were localized to the chromosome 19 physical map.

Depending on the ionic environment the replicative complex of silkworm Bombyx mori, containing DNA polymerase alpha and primase, catalyzes on single-stranded DNA of phage M13 a NTP-dependent synthesis or elongation of preformed primers. In the presence of NTPs and dNTPs at conditions optimal for the NTP-dependent synthesis the replicative complex synthesizes on M13 DNA oligoribonucleotides of 9-11 residues, which serve as primers for polymerization of DNA. The length of RNA-primers synthesized by primase of the complex depends on concentration of dNTP but does not depend on activity of DNA polymerase alpha. During elongation of exogenic primers annealed to M13 DNA the complex is processive synthesizing DNA fragments of dozens residues without dissociation from the template. Double-stranded structures in DNA such as "hairpins" appear to be barriers for driving of the complex along the template and cause pauses in elongation. DNA-binding proteins the SSB of Escherichia coli or the p32 of phage T4 destabilize double-stranded regions in DNA and eliminate elongation pauses corresponding to these regions. The replicative complex is able to fill in single-stranded gaps in DNA completely and to perform slowly the synthesis with displacement of one of parent strands in duplexes via repeated cycles of binding to the primer-template, limited elongation and dissociation.

Induction of heat shock proteins (HSP's) synthesis in human lymphocytes in vitro was studied using 1D- and 2D-polyacrylamide gel electrophoresis. High resolution of the O'Farrell's procedure revealed the existence of several isoforms of HSP70. The same major HSP's (70 kDa and 88 kDa) were found in rabbit lymphocytes in vivo under whole body hyperthermia. Freshly isolated lymphocytes seem to offer a new approach to the investigation of HSP synthesis induction in mammals under whole body hyperthermia.

Previously, using a cell culture system we have shown that heat shock proteins (HSPs) transported to the nucleus were revealed in association with the nuclear matrix (Akopov et al., 1986; Buldyaeva et al., 1986). We have found as well that whole body heating of rats induced alteration of liver nuclear matrix protein metabolism similar to that in cell culture under heat shock (Akopov et al., 1985). It is known that HSP synthesis leads to thermotolerance i.e. thermoresistancy of organisms or cells to usually lethal heating.