M. B. Kostina

Ouabain-sensitive H,K-ATPase: tissue-specific expression of the mammalian genes encoding the catalytic α subunit1

Human ATP1AL1 and corresponding genes of other mammals encode the catalytic α subunit of a non‐gastric ouabain‐sensitive H,K‐ATPases, the ion pump presumably involved in maintenance of potassium homeostasis. The tissue specificity of the expression of these genes in different species has not been analyzed in detail. Here we report comparative RT‐PCR screening of mouse, rat, rabbit, human, and dog tissues. Significant expression levels were observed in the skin, kidney and distal colon of all species (with the exception of the human colon). Analysis of rat urogenital organs also revealed strong expression in coagulating and preputial glands. Relatively lower expression levels were detected in many other tissues including brain, placenta and lung. In rabbit brain the expression was found to be specific to choroid plexus and cortex. Prominent similarity of tissue‐specific expression patterns indicates that animal and human non‐gastric H,K‐ATPases are indeed products of homologous genes. This is also consistent with the high sequence similarity of non‐gastric H,K‐ATPases (including partial sequences of hitherto unknown cDNAs for mouse and dog proteins).

Structural evolution and tissue-specific expression of tetrapod-specific second isoform of secretory pathway Ca2+-ATPase.

Secretory pathway Ca-ATPases are less characterized mammalian calcium pumps than plasma membrane Ca-ATPases and sarco-endoplasmic reticulum Ca-ATPases. Here we report analysis of molecular evolution, alternative splicing, tissue-specific expression and subcellular localization of the second isoform of the secretory pathway Ca-ATPase (SPCA2), the product of the ATP2C2 gene. The primary structure of SPCA2 from rat duodenum deduced from full-length transcript contains 944 amino acid residues, and exhibits 65% sequence identity with known SPCA1. The rat SPCA2 sequence is also highly homologous to putative human protein KIAA0703, however, the latter seems to have an aberrant N-terminus originating from intron 2. The tissue-specificity of SPCA2 expression is different from ubiquitous SPCA1. Rat SPCA2 transcripts were detected predominantly in gastrointestinal tract, lung, trachea, lactating mammary gland, skin and preputial gland. In the newborn pig, the expression profile is very similar with one remarkable exception: porcine bulbourethral gland gave the strongest signal. Upon overexpression in cultured cells, SPCA2 shows an intracellular distribution with remarkable enrichment in Golgi. However, in vivo SPCA2 may be localized in compartments that differ among various tissues: it is intracellular in epidermis, but enriched in plasma membranes of the intestinal epithelium. Analysis of SPCA2 sequences from various vertebrate species argue that ATP2C2 gene radiated from ATP2C1 (encoding SPCA1) during adaptation of tetrapod ancestors to terrestrial habitats.

Genetic markers for lung and esophagus common precursor cells in human development

203 It is known that cancer often shows signs of “embryonization”—phenotypic characteristics of those stem or progenitor cells that are the primary site of origin of a particular organ. This phenomenon, sometimes referred to as “persistence of embryonic traits,” is associated with the fact that the change in the cell phenotype from the normal to tumor involves the same signal and regulatory systems that in the nor mal embryonic development of this tissue. In this con nection, this feature can be used to obtain information about the molecular processes of normal develop ment, especially at the early stages, when an organ is composed of a small number of cells and the very pro cess of its transition to another stage of development is too rapid.

KLF5, a new player and new target in the permanently changing set of pancreatic cancer molecular drivers

Pancreatic cancer is one of the most aggressive tumor types characterized by chemotherapy resistance and high metastatic activity. Recent studies revealed new genes, which are likely to be actively involved in the regulation of the processes occurring in the pancreas, as well as in the development of cancer in this organ. This review is devoted to the description of one of the recently revealed genes, KLF5, which seems to be a promising target for therapeutic intervention in the most widespread type of pancreatic cancer, ductal adenocarcinoma.

Contiguous strings of strongly binding short oligonucleotides as a useful tool for completing sequencing experiments.

Large-scale DNA sequencing is currently based mostly on the shotgun approach. Although widely used, it is not free of shortcomings: the total length of randomly sequenced subclones appear to be five to eight times more than the total DNA length; such a high statistical redundancy of random sequences cannot guarantee the absence of unsequenced gaps. We calculated that these problems could be alleviated if the average length of the subcloned random fragments were increased. In this paper we present the primer walking approach based on the use of contiguous modified hexamer strings strongly binding to complementary templates as segmented primers. The approach exhibits the following characteristic features: 1) Single-stranded templates appear to have advantage over double-stranded ones. 2) With single-stranded templates, an overall sequencing success rate is about 80%. 3) A relatively small library of 1400 hexanucleotides selected according to the most frequent occurrence in the human genome is sufficient for sequencing all clones. The approach allows one to reduce the number of templates necessary for the shotgun strategy and also to decrease the number of gaps between sequenced contigs. This should be kept in mind when considering the ways for enhancement of the present strategy of large-scale sequencing.

Tissue Specificity of Alternative Splicing of Transcripts Encoding Hampin, a New Mouse Protein Homologous to the Drosophila MSL-1 Protein

A number of mammalian genomes have one gene copy encoding the protein that we named hampin. A search in a number of databases revealed a distant homologue, the well-known Drosophila protein MSL-1 (male-specific lethal 1). An alternative splicing of mRNA led to a significant diversity of structural hampin variants with different domain compositions. We analyzed the tissue-specific expression of five mouse hampin variants using RT-PCR. Two variants encoding hampin proteins with truncated N termini were shown to have a restricted tissue specificity: they are exclusively expressed in the testes. The mRNAs of other hampin variants were detected in all the tested tissues at comparable levels. We obtained polyclonal antibodies to the recombinant hampin and used them to demonstrate that at least one of the variants is predominantly localized in the nucleus. The specific features of the hampin primary structure and its possible functions as a member of the hampin/MSL-1 family of proteins are discussed.

The Transcription Activation of the RIG-I Gene Encoding the DEXH/D Protein in RH Cells Infected with Tick-Borne Encephalitis Virus

It was demonstrated by subtractive hybridization that the infection of a human embryonic kidney cell line with tick-borne encephalitis virus causes an approximately tenfold transcription activation of the RIG-I gene, which encodes a protein of the DEXH/D-box-containing RNA helicase family. A possible involvement of the protein in antiviral cell systems is discussed.

CRISPR/CAS Targeted in vivo Genome Modification for Studying the Functional Role of Genomic Regulatory Elements in Health and Carcinogenesis

One problem in the study of regulatory mechanisms in living systems is the difficulty in analysis of regulatory elements in their natural context. One promising way to solve this problem is direct in situ modification of regulatory element sequences. The new technology of gene modification based on the bacterial CRISPR/Cas system allows one to quickly and accurately modify any genomic fragment, in particular, in cells of an adult organism. This review considers principles of the CRISPR/Cas technology and its application to the study of regulatory elements, as well as the potential of using this technology in studies of regulatory systems in pancreas tumors.

Antitumor efficacy of combined gene and radiotherapy in animals

Antitumor efficacy of the combined suicide gene therapy and radiotherapy was studied on the model of CT26 murine colon adenocarcinoma. CMV-FCU1-IRES-mGM-CSF-pGL3 construct with PEG-PEI-TAT (FCU1–mGM/5-FC) block copolymer as a vector was used for intratumoral administration. Tumors were irradiated with a single 5 Gy dose. The efficacy was evaluated according to the grade of tumor growth inhibition (T/C) and lifespan of the animals. Pronounced antitumor activity of the combined use of FCU1–mGM/5-FC system with radiotherapy on the background of prolonged lifespan and the synergism of the applied methods was revealed.

Expression of Therapeutic Gene FCU1 Sensitizes Pancreatic Cancer Cells to 5-Fluorocytosine and Enhances the Cytotoxic Effect of 5-Fluorouracil

Hybrid therapeutic gene FCU1 gene was cloned into a lentiviral expression vector and the therapeutic effect of its expression was studied in three pancreatic cancer cell lines. Expression of FCU1 gene sensitized cells of two of three studied pancreatic cancer cell lines to 5-fluorocytosine. In addition, uracil phosphoribosyl transferase activity of the hybrid FCU1 protein increased sensitivity of transfected cells of all three studied pancreatic cancer cell lines to 5-fluorouracil, a standard chemotherapeutic agent.