[A novel approach to identification of somatic retroelements' insertions in human genome].

The activity of retroelements is one of the factors leading to genetic variability of the modern humans. Insertions of retroelements may result in alteration of gene expression and functional diversity between cells. In recent years an increasing amount of data indicating an elevated level of retroelements' mobilisation in some human and animal tissues has been reported. Therefore, the development of a system for the detection of somatic retroposition events is required. Here we describe a novel approach to the whole-genome identification of somatic retroelemts' insertions in human genome. The developed approach was applied for the comparisons of somatic mosaicism levels in two tissues of the investigated individual. A total of 3410 insertions of retroelements belonging to AluYa5 subfamily were identified.

[The functional analysis of polymorphic insertions of Alu retroelements at acute lymphoblastic leukemia].

Human genome variability observed within patient cohorts is considered as a goal of functional genomics essential for personalized medicine progress. In the current research we implement functional analysis of 31 polymorphic Alu insertions located within gene introns for individual genomes of patients with acute lymphoblastic leukemia (ALL). As a result we demonstrated a decrease of the primary transcripts content for 21 Alu-containing alleles. The most strong inhibitory effect of 10 Alu insertions was observed in both mononuclear blood cells of healthy donors and B-lymphoblasts of ALL patients. Allele frequencies of three Alu insertions that are located in MEF2C (two of them) and TAX1BP1 genes significantly differ (p-value 0.027. 0.052. 0.014 accordingly) between cohorts of healthy donors and ALL patients. Prolong influence of the Alu insertions on intracellular content of mature mRNA was studied for corresponding allele of TARBP1 gene.

[The oligoclonal expansion of T cells: study of its stability over time].

A novel experimental approach to the investigation of the repertoire of peripheral T lymphocytes of patients suffering from ankylosing spondylitis (AS) is proposed. This approach is based on the wide-range sequencing of cDNA of the beta-chain of the T-cellular receptor (TcR). The results of the analysis of the diversity of sequences of the TcR antigen-binding domain (CDR3) inside the total pool of one patient with AS are presented by the example of the second V family (BV2) of TcR. The expansion of six independent TcR-expressing clones of T cells with a similar amino acid sequence of the CDR3 domains was proposed based on the results of the comparative structural analysis of the clone libraries of the cDNA of TcR BV2. The long-time stable expansion of these T clones was demonstrated during the development of the disease by specific monitoring.

[Retroposons in modern human genome evolution].

The ascertainment of the rates and driving forces of human genome evolution along with the genetic diversity of populations or separate population groups remains a topical problem of fundamental and applied genomics. According to the results of comparative analysis, the most numerous human genome structure peculiarities are connected with the distribution of mobile genetic retroelements - LTR, LINE1, SVA, and Alu repeats. Due to the wide distribution in different genome loci, conversed retropositional activity, and the retroelements regulatory potential, let us regard them as one of the significant evolutionary driving forces and the source of human genome variability. In the current review, we summarize published data and recent results of our research aimed at the analysis of the evolutionary impact of the young retroelements group on the function and variability of the human genome. We examine modern approaches of the polygenomic identification of polymorphic retroelements inserts. Using an original Internet resource, we analyze special features of the genomic polymorphic inserts of Alu repeats. We thoroughly characterize the strategy of large-scale functional analysis of polymorphic retroelement inserts. The presented results confirm the hypothesis of the roles of retroelements as active cis regulatory elements that are able to modulate surrounding genes.

[A new database on polymorphic retroelements in human genome (PRED)].

Comparison of primate genomes sequences has confirmed the evidence that substantial part of intra- and interspecies differences is provided by retroelements. Human genome contains thousands of polymorphic retroelement copies considered to be perspective molecular genetic markers of new generation. However utilization of polymorphic retroelements as molecular genetic markers is limited due to lack of systematic data on their number, genomic context and distribution among human populations. We have created first bilingual (Russian/English) internet-resource devoted to known polymorphic retroelements discovered in human genome by our group as well as by other researchers worldwide. The database contains information about each retroelement copy location, position relative to known and predicted genes, frequency of alleles in human populations and others. Our internet portal allows to perform a search in database using multiple search conditions and available on http://labcfg.ibch.ru/home.html. The database provides an opportunity to investigate distribution of polymorphic retroelements in human genome and to design new genetic markers for various population and medical studies.

[A tissue-specific decrease in the pre-mRNA level of L1- and alu-containing alleles of human genes].

LINE1 and Alu retroelements occupy approximately 17 and 13% of the human genome, respectively. They include the evolutionarily youngest element groups Ta-L1, AluYa5, and AluYb8, many inserts of which are polymorphous in the Homo sapiens population. Despite the data on the ability of L1 and Alu elements to cause various modifications of the genome, the effects of these retroelements on gene expression have yet not been studied. Using the RT PCR method, we analyzed the pre-mRNA (heterogeneous nuclear RNA) content of allele pairs of four genes in five human cell lines, heterozygous with respect to intronic inserts of L1 and Alu elements. We showed for the first time a tissue-specific decrease in the pre-mRNA content of the gene allele bearing L1 or Alu inserts relative to the other allele of the same gene lacking the retroelement.

[The tissue-specific methylation of human-specific endogenous retroviral long terminal repeats]. / Tkanespetsifichnoe metilirovanie spetsifichnykh dlia cheloveka dlinnykh kontsevykh povtorov éndogennykh retrovirusov.

A possible involvement of retroelements in the epigenetic regulation of human gene expression was considered by the example of methylation of long terminal repeats (LTRs) of the human endogenous retrovirus family K (HERV-K). The methylation status of six HERV-K LTRs was determined in various gene-enriched regions of the human genome. The methylation of four LTRs was shown to be tissue-specific. Our results correlated with published data on the tissue-specific changes in the expression level of human genes adjacent to the LTRs under study. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 5; see also http: // www.maik.ru.

[Genetic structure of people from the Volga-Ural region and Central Asia from data of Alu-polymorphism]. / Geneticheskaia struktura narodov volgo-ural'skogo regiona i srednei azii po dannym Alu-polimorfizma.

Nine Alu loci (Ya5NBC5, Ya5NBC27, Ya5NBC148, Ya5NBC182, YA5NBC361, ACE, ApoA1, PV92, TPA25) were analyzed in six ethnic populations (Trans-Ural Bashkirs, Tatars-Mishars, Mordovians-Moksha, Mountain Maris, Udmurts, and Komi-Permyaks) of the Volga-Ural region and in three Central Asian populations (Uzbeks, Kazakhs, and Uigurs). All Alu insertions analyzed appeared to be polymorphic in all populations examined. The frequency of insertion varied from 0.110 in Mountain Maris at the Ya5NBC5 locus to 0.914 in Tatars at the ApoA1 locus. The data on the allele frequency distribution at nine loci point to the existence of substantial genetic diversity in the populations examined. The value of the observed heterozygosity averaged over nine Alu insertions varied from 0.326 in Mountain Maris to 0.445 in Kazakhs and Uigurs. The level of the interpopulation genetic differences for the Volga-Ural population (Fst = 0.061) was higher than for the populations of Central Asia (Fst = 0.024), Europe (Fst = 0.02), and Southeastern Asia (Fst = 0.018). The populations examined were highly differentiated both in respect of linguistic characteristics and the geographical position. The data obtained confirmed the effectiveness of the marker system used for the assessment of genetic differentiation and the relationships between the ethnic groups.

[Nucleotide sequences of long terminal repeats of the human endogenous retrovirus (LTR HERV-K) on the short arm of chromosome 7: identification, analysis and evaluation of transcriptional activity]. / Posledovatel'nosti dlinnykh kontsevykh povtorov éndogennogo retrovirusa (LTR HERV-K) korotkogo plecha khromosomy 7 cheloveka: poisk, analiz i opredelenie transkriptsionnoi aktivnosti.

Six clones containing long terminal repeat (LTR) sequences of human endogenous retrovirus of the HERV-K family were found in the YAC library (1200 kb) of the short arm of human chromosome 7. The sequence sizes of the three clones corresponded to the full-length LTR (969 bp). The LTR localization was determined using FISH and verified by comparison with the GenBank database. All three DNA fragments containing solitary LTRs were transcribed in normal germline cells (testicular parenchyma tissue). The differences in the expression of these clones in the germline tumor cells (seminoma) were observed.

[Human genome-specific HERV-K intron LTR genes have a random orientation relative to the direction of transcription, and, possibly, participated in antisense gene expression regulation]. / Spetsifichnye dlia genoma cheloveka LTR HERV-K v intronakh genov imeiut nesluchainuiu orientatsiiu otnositel'no napravleniia transkriptsii i, vozmozhno, prinimaiut uchastie v antisens-reguliatsii ékspressii genov.

A consensus nucleotide sequence of long terminal repeats (LTRs) of endogenous human-specific retroviruses of the K family (HERV-K) was constructed and used for the genome-wide search for homologies in international databases. There were revealed 142 LTRs, 12 of which were localized in introns of unique human genes. It was found for the first time that ten intron LTRs are absent in the orthologic loci of the chimpanzee genome and the orientation of nine of them is opposite to the transcription direction of the corresponding human genes. A hypothesis was propounded that the found LTRs affect the gene expression by initiation of the antisense RNA synthesis.

[Enhancer activity of solitary long terminal repeat of the human endogenous retrovirus of the HERV-K family]. / Enkhansernaia aktivnost' vnevirusnogo dlinnogo kontsevogo povtora éndogennogo retrovirusa cheloveka semeistva HERV-K.

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.

[New putative gene from human retrovirus-containing locus on chromosome 19]. / Novyi potentsial'nyi gen is retrovirusosoderzhashchego lokusa 19-i khromosomy cheloveka.

By means of cDNA selection, a cDNA clone corresponding to a putative gene was isolated. The identified gene was mapped to human chromosome 19 (region 19q12) to the locus containing the human endogenous retrovirus HERV-K LTR. The distance between the LTR and the cDNA fragment is about 20 kb. RT-PCR analysis of the total RNA revealed that the corresponding gene is preferentially expressed in the brain.

[Long terminal repeat of the human endogenous retrovirus HERV-K in the intron of the ZNF91 gene]. / Dlinnyi kontsevoi povtor éndogennogo retrovirusa cheloveka HERV-K v introne gena ZNF91.

The relative positions of the ZNF91 gene (exon-intron organization) and the HERV-K human endogenous virus long terminal repeat (LTR), which was earlier found to be located in the ZNF91 gene locus, on the EcoRI restriction metric map of the human chromosome 19 were determined with a high resolution. The direction of the ZNF91 gene transcription relative to the chromosome 19 telomeres was determined. The HERV-K LTR was localized to the ZNF91 gene intron 19. The role of retroviral sequences in the evolution of the ZNF91 gene family is discussed.

[Structural characteristics of four long terminal repeats (LTR) of human endogenous retroviruses and features of their integration sites]. / Strukturnye kharakteristiki chetyrekh dlinnykh kontsevykh povtorov (LTR) endogennykh chelovecheskikh virusov i osobennosti uchastkov ikh integratsii.

Four LTR-containing regions of human chromosome 19 were sequenced by the primer walking technique using strings of short oligonucleotides tightly bound to the template. A comparative and evolutionary analysis of sequences homologous to human endogenous retroviruses (HERV) was performed, and the prototypes of the LTRs were determined. Analysis of the chromosome 19 sequences adjacent to LTR revealed that LTRs of HERV-K share a common location with other retroposons.

[Isolation and characteristics of an ordered library of transcribed sequences of human chromosome 19 from hybrid human-hamster cells]. / Poluchenie i kharakteristiki uporiadochennoi biblioteki transkribiruemykh posledovatel'nostei khromosomy 19 cheloveka iz gibridnykh kletok chelovek-khomiak.

A new technique based on Alu-PCR amplification of hn-RNA is described for the extraction of human-specific transcribed sequences from a hybrid cell line. Arrayed library of hn-cDNA was constructed and characterized by sequencing about 80 individual clones. A high enrichment by human-specific sequences (about 95%) was demonstrated.