[Hsp70 Genes of the Megaphragma amalphitanum (Hymenoptera: Trichogrammatidae) Parasitic Wasp].

Miniaturization is an evolutionary process that is widely represented in both invertebrates and vertebrates. Miniaturization frequently affects not only the size of the organism and its constituent cells, but also changes the genome structure and functioning. The structure of the main heat shock genes (hsp70 and hsp83) was studied in one of the smallest insects, the Megaphragma amalphitanum (Hymenoptera: Trichogrammatidae) parasitic wasp, which is comparable in size with unicellular organisms. An analysis of the sequenced genome has detected six genes that relate to the hsp70 family, some of which are apparently induced upon heat shock. Both induced and constitutively expressed hsp70 genes contain a large number of introns, which is not typical for the genes of this family. Moreover, none of the found genes form clusters, and they are all very heterogeneous (individual copies are only 75-85% identical), which indicates the absence of gene conversion, which provides the identity of genes of this family in Drosophila and other organisms. Two hsp83 genes, one of which contains an intron, have also been found in the M. amalphitanum genome.

Pleiotropic effect of thyroid hormones on gene expression in fish as exemplified from the blue bream Ballerus ballerus (Cyprinidae): Results of transcriptomic analysis.

A pronounced pleiotropic effect of thyroid hormones on the regulation of gene expression in fish in postembryogenesis was demonstrated for the first time using larvae and juveniles of the blue bream Ballerus ballerus as an example. Genome-wide transcriptome sequencing (RNA-seq) identified 1212 differentially expressed genes in the brain and liver of fish kept in triiodothyronine solution (0.25 ng/mL). Our data show that the regulation of gene expression by thyroid hormones is widespread in nature: it involves not only the structural genes but also the regulatory genes. A significant number of genes under the control of thyroid hormones are involved in the determination of morphological traits.

Genomic Study of Cardiovascular Continuum Comorbidity.

Comorbidity or a combination of several diseases in the same individual is a common and widely investigated phenomenon. However, the genetic background for non-random disease combinations is not fully understood. Modern technologies and approaches to genomic data analysis enable the investigation of the genetic profile of patients burdened with several diseases (polypathia, disease conglomerates) and its comparison with the profiles of patients with single diseases. An association study featuring three groups of patients with various combinations of cardiovascular disorders and a control group of relatively healthy individuals was conducted. Patients were selected as follows: presence of only one disease, ischemic heart disease (IHD); a combination of two diseases, IHD and arterial hypertension (AH); and a combination of several diseases, including IHD, AH, type 2 diabetes mellitus (T2DM), and hypercholesterolemia (HC). Genotyping was performed using the "My Gene" genomic service (www.i-gene.ru). An analysis of 1,400 polymorphic genetic variants and their associations with the studied phenotypes are presented. A total of 14 polymorphic variants were associated with the phenotype "IHD only," including those in the APOB, CD226, NKX2-5, TLR2, DPP6, KLRB1, VDR, SCARB1, NEDD4L, and SREBF2 genes, and intragenic variants rs12487066, rs7807268, rs10896449, and rs944289. A total of 13 genetic markers were associated with the "IHD and AH" phenotype, including variants in the BTNL2, EGFR, CNTNAP2, SCARB1, and HNF1A genes, and intragenic polymorphisms rs801114, rs10499194, rs13207033, rs2398162, rs6501455, and rs1160312. A total of 14 genetic variants were associated with a combination of several diseases of cardiovascular continuum (CVC), including those in the TAS2R38, SEZ6L, APOA2, KLF7, CETP, ITGA4, RAD54B, LDLR, and MTAP genes, along with intragenic variants rs1333048, rs1333049, and rs6501455. One common genetic marker was identified for the "IHD only" and "IHD and AH" phenotypes: rs4765623 in the SCARB1 gene; two common genetic markers, rs663048 in SEZ6L and intragenic rs6501455, were identified for the "IHD and AH" phenotype and a combination of several diseases (syntropy); there were no common genetic markers for the "syntropy" and "IHD only" phenotypes. Classificatory analysis of the relationships between the associated genes and metabolic pathways revealed that lipid-metabolizing genes are involved in the development of all three CVC variants, whereas immunity-response genes are specific to the "IHD only" phenotype. The study demonstrated that comorbidity presents additional challenges in association studies of disease predisposition, since the genetic profile of combined forms of pathology can be markedly different from those for isolated "single" forms of a disease.

[Bifunctional role of domain zinc fingers of methyl-DNA-binding protein Kaiso].

DNA methylation in mammals is one of the major epigenetic mark that associates with inactive chromatin state. Methyl-DNA-binding proteins bind methylated DNA and silence gene transcription by recruiting repression complexes. Kaiso is one of the methyl-DNA-binding proteins. It has a domain structure: N-terminal BTB/POZ domain involved in protein-protein interaction and C-terminal zinc-fingers of C2H2 type that bind methylated DNA (mCGmCG) or nonmethylated - TCCTGCNA. Here we show that Kaiso interacts with p120 catenin through zinc finger 2 and 3. This interaction has dual consequences. Firstly, binding to p120 inhibits nuclear import of Kaiso that results in most of Kaiso-p120 complexes becoming cytoplasmic. And secondly, bound p120 makes impossible interaction of the zinc fingers with methylated DNA. These modes of Kaiso modulation by p120 can open attractive perspectives in linking events on cell membrane and changes in nuclear gene expression.

[Transcription factor ZF5 regulates expression of mammalian gene containing GCC-triplet repeats in 5'-regulatory region in human hepatoma HepG2 cells].

Some nuclear proteins of human HeLa and HepG2 cells are capable of binding to GCC-triplet repeats--(GCC)n > 3 in 5'-regulatory regions of a number of mammalian genes--G-C-elements. According to our previous data, nucleotide sequence (GCC)4 in promoter of mouse ribosomal protein L32 gene (rpL32) between 17 and 6 bp upstream of transcription start site interacts to nuclear proteins from HepG2 cells, and may be considered as a GCC-element. We suggest that one of those proteins, with molecular weight about 52 kDa, which may interact with rpL32 GCC-element, is a known conservative mammalian transcription factor ZF5. DNA-binding domain of ZF5 contains a few Kruppel-like Zn-fingers (Cys2His2-type) interacting with the GC-rich nucleotide sequences in 5'-regulatory regions of a number of mammalian genes. Our results (obtained by EMSA) showed that recombinant GST-ZF5 fused protein containing ZF5 DNA-binding domain specifically binds a few GS-rich sequences: (GCC)g-9riplet repeats, 5'-GCGCGC-3' (known ZF5 consensus binding site) and (more preferable) the fragment (-24...+1 bp) of rpL32 promoter. The high affinity of ZF5 DNA-domain binding with the latter may be explained by the presence in this fragment of two overlapped subsequences, each being capable of binding to ZF5: (GCC)4 and 5'-GCGCGC- 3'. Zf5 cDNA was cloned from HepG2 cells by RT-PCR method, and then used for construction of the gene expression vector. It has been shown that Zf5 cDNA expression vector specifically down-regulates (in luciferase assays) the activity of rpL32 promoter (-155...+159) including the above mentioned GC-rich subsequences by cotransfection of HepG2 cells. Therefore, our results enable us to consider GCC-elements as a novel class of ZF5 targets in 5'-regulatory regions of mammalian genes.

Methylation of DNA--one of the major epigenetic markers.

Regulation of gene expression is a complex process. It includes a great number of steps from control of mRNA synthesis to posttranslational modification of proteins. Epigenetic events play essential roles in regulation of transcription. In this review, we concentrate on methylation of DNA as one of the important epigenetic marks. It is well known that DNA methylation is associated with closed chromatin state and, therefore, repressed, inactive genes. Here we describe major processes that depend on DNA methylation: imprinting, X-inactivation, and oncogenesis. Also we describe a number of known methyl-DNA-binding proteins and links between methylation of DNA and higher-order chromatin structure.

[KAISO--a new member of the BTB/POZ family specifically bindsto methylated DNA sequences]. / KAISO, novyi chlen VTV/POZ semeistva, spetsifichno sviazyvaetsia s metilirovannymi posledovatel'nostiami DNK.

A protein specifically binding a symmetrically methylated DNA fragment of the first intron of the mts1 gene was studied. The protein was purified by gel filtration and affinity chromatography. Mass spectrometry showed that the protein is Kaiso, a new member of the BTB/POZ family. To study the association with methylated DNA sequences in vivo, the location of Kaiso in NIH 3T3 cells was analyzed. Immunofluorescent staining with polyclonal antibodies against Kaiso showed that the protein is predominantly associated with the nucleoli. The causes of its distribution awaits further investigation. The zinc-finger domains of Kaiso were for the first time demonstrated to specifically recognize symmetrically methylated DNA sequences in vitro.

[Functional characteristics of the promoter region of the tag7/PGRP gene in KSML0, KSML100 murine mammary adenocarcinoma cell ines and VMR liver]. / Funktsional'naia kharakteristika promotornoi oblassti gena tag7/PGRP v kletkakh linii KSML0, KSML100 i VMR-pechen' adenokartsinom molochnoi zhelezy myshi.

A study was carried out on the transcription regulation of the tag7/PGRP gene, whose product is similar to cytokines and is involved in recognizing peptidoglycane in mouse mammary adenocarcinoma cell lines KSML0, KSML100, and VMR-liver. The 3250-bp fragment of the promoter region was sequenced and tested for DNase I-hypersensitive sites (DHSs). In the KSML0 cells line, DHSs were found in the vicinity of the TATA box and approximately at position -3000 relative to the transcription start of the gene. Only a DHS cluster near the TATA box was found in the VMR-liver cell line. Regions involved in transcription regulation of the gene in the three cell lines were identified with the use of reporter constructions carrying the CAT gene under the control of deletion derivatives of the tag7/PGRP promoter region. The minimal promoter including only the TATA box with the nearest neighboring sequences was inactive in all cell lines. The elements (enhancers) positively regulating gene transcription in KSML0 and VMR-liver were mapped to region -192, -48. An element accounting for the transcriptional inactivity of the gene in KSML100 cells was assigned to region -315, -3250.

[Analysis of homologous DNA sequences within the first intron of the mouse and human mts1 genes: kB-like site and microsatellite DNA]. / Analiz gomologichnykh posledovatel'nostei DNK v pervom introne gena mts1 myshi i cheloveka: kB-podobnyi sait i mikrosatellitnaia DNK.

Homologous regions were revealed and analyzed within the first intron of the mouse and human mts1 genes. Maximal homology was detected between microsatellite DNA sequences from the first intron of the mouse gene (+804, +863) and the human gene (+600, +645). DNase I hypersensitive sites were revealed within these regions in both mouse and human cell lines tested, thus showing functional significance of the homology detected. In both the mouse and the human genes, the 5' end of homologous regions is flanked by a DNA sequence similar to a NF-kB/Rel protein-binding consensus sequence. Previously, this sequence was demonstrated to be involved in a complex regulatory element of the mouse mts1 gene. In comparison with the kB-like sequence, mouse, rat, and human sequences were found to involve one, two, and three nucleotide substitutions, respectively. Alterations in a spectrum of nuclear proteins bound to various kB-like sequences were analyzed. Possible effects of these alterations on mts1 transcription regulation were discussed. The possibility of the human kB-like sequence to function as positive regulator of transcription initiation was demonstrated in vivo.