V. Z. Tarantul

Mitochondrial 16S rRNA gene encodes a functional peptide, a potential drug for Alzheimer’s disease and target for cancer therapy

New functions of well-known genes have been revealed frequently. A new example is described in this report. Earlier we have detected an up-regulation of expression of the mitochondrial 16S rRNA gene in non-Hodgkins lymphomas. Here we demonstrate that the human mitochondrial 16S rRNA gene encodes a potential oncopeptide, Humanin described recently. This peptide suppresses neuronal cell death induced by mutant genes responsible for familial Alzheimers disease (AD). Analysis of the gene coding site structure showed that Humanin mRNA is translated most likely in the cytosol, but not in the mitochondrion in vivo. This led us to suppose that AD could be caused by a block of Humanin mRNA transport from mitochondria into the cytosol. Moreover, our data and reports by others an mitochondrial 16S rRNA transcription and characteristic of transcript structure suggests that Humanin is a potential oncopeptide. Thus, the use of Humanin for the treatment of AD may increase the risk for the development of malignant diseases.

Differential gene expression in B-cell non-Hodgkin's lymphoma of SIV-infected monkey

Infection with SIVmac251 in some rhesus monkeys (Macaca mulatta) leads to B-cell non-Hodgkins lymphomas (B-NHL) clinically similar to that of HIV-infected AIDS patients. To further characterize the SIV-associated B-NHL we have generated genetic profiles of malignant cells by subtractive hybridization and Northern blot analysis. We have analyzed 21 clones of a subtracted cDNA library corresponding to overexpressed genes in diffuse large B-cell (DLBCL) SIV-associated monkey lymphoma. Eight of these clones represent a sequence homologous to an abundant transcript from KG-1 cells originally established from a human myelogenous leukemia. The protein encoded has a 60% similarity to a hypothetical glycine-rich transmembrane signal protein of Caenorhabditis elegans and 25% similarity to the ret finger protein. The other cDNA clones contained sequences of the serum amyloid A gene (SAA), the alpha1-acid glycoprotein gene (AGP), the ribosomal protein S3a (RPS3a) and L8 (RPL8) genes, the interferon-inducible gene (INF-ind), the metastasin gene (mts1), and the NADH dehydrogenase I gene (ND-I). The remaining cDNA clones consisted of yet unknown sequences. In addition, we detected an up-regulation of the cytochrome c oxidase II gene (COX-II), the ND-IV gene, and the SET oncogene by Northern blot hybridization in three SIV-associated NHLs of different histomorphological classification. All these genes have not previously been found to be overexpressed in B-NHL.

Inaccurate DNA Synthesis in Cell Extracts of Yeast Producing Active Human DNA Polymerase Iota

Mammalian Pol ι has an unusual combination of properties: it is stimulated by Mn2+ ions, can bypass some DNA lesions and misincorporates “G” opposite template “T” more frequently than incorporates the correct “A.” We recently proposed a method of detection of Pol ι activity in animal cell extracts, based on primer extension opposite the template T with a high concentration of only two nucleotides, dGTP and dATP (incorporation of “G” versus “A” method of Gening, abbreviated as “misGvA”). We provide unambiguous proof of the “misGvA” approach concept and extend the applicability of the method for the studies of variants of Pol ι in the yeast model system with different cation cofactors. We produced human Pol ι in bakers yeast, which do not have a POLI ortholog. The “misGvA” activity is absent in cell extracts containing an empty vector, or producing catalytically dead Pol ι, or Pol ι lacking exon 2, but is robust in the strain producing wild-type Pol ι or its catalytic core, or protein with the active center L62I mutant. The signature pattern of primer extension products resulting from inaccurate DNA synthesis by extracts of cells producing either Pol ι or human Pol η is different. The DNA sequence of the template is critical for the detection of the infidelity of DNA synthesis attributed to DNA Pol ι. The primer/template and composition of the exogenous DNA precursor pool can be adapted to monitor replication fidelity in cell extracts expressing various error-prone Pols or mutator variants of accurate Pols. Finally, we demonstrate that the mutation rates in yeast strains producing human DNA Pols ι and η are not elevated over the control strain, despite highly inaccurate DNA synthesis by their extracts.

A false note of DNA polymerase iota in the choir of genome caretakers in mammals

DNA polymerase iota (Polι) of mammals is a member of the Y family of DNA polymerases. Among many other genome caretakers, these enzymes are responsible for maintaining genome stability. The members of the Y-family DNA polymerases take part in translesion DNA synthesis, bypassing some DNA lesions, and are characterized by low fidelity of DNA synthesis. A unique ability of Polι to predominantly incorporate G opposite T allowed us to identify the product of this enzyme among those synthesized by other DNA polymerases. This product can be called a “false note” of Polι. We measured the enzyme activity of Polι in crude extracts of cells from different organs of five inbred strains of mice (C3H/Sn, 101/H, C57BL/6, BALB/c, 129/J) that differed in a number of parameters. The “false note” of Polι was clearly sounding only in the extracts of testis and brain cells from four analyzed strains: C3H/Sn, 101/H, C57BL/6, BALB/c. In mice of 129/J strain that had a nonsense mutation in the second exon of the polι gene, the Polι activity was reliably detectable only in the extracts of brain. The data show that the active enzyme can be formed in some cell types even if they carry a nonsense mutation in the polι gene. This supports tissue-specific regulation of polι gene expression through alternative splicing. A semiquantitative determination of Polι activity in mice strains different in their radiosensitivity suggests a reciprocal correlation between the enzyme activity of Polι in testis and the resistance of mice to radiation.

Effect of human cell malignancy on activity of DNA polymerase ι

An increased level of mutagenesis, partially caused by imbalanced activities of error prone DNA polymerases, is a key symptom of cell malignancy. To clarify the possible role of incorrect DNA polymerase ι (Pol ι) function in increased frequency of mutations in mammalian cells, the activity of this enzyme in extracts of cells of different mouse organs and human eye (melanoma) and eyelid (basal-cell skin carcinoma) tumor cells was studied. Both Mg2+, considered as the main activator of the enzyme reaction of in vivo DNA replication, and Mn2+, that activates homogeneous Pol ι preparations in experiments in vitro more efficiently compared to all other bivalent cations, were used as cofactors of the DNA polymerase reaction in these experiments. In the presence of Mg2+, the enzyme was active only in cell extracts of mouse testicles and brain, whereas in the presence of Mn2+ the activity of Pol ι was found in all studied normal mouse organs. It was found that in cell extracts of both types of malignant tumors (basal-cell carcinoma and melanoma) Pol ι activity was observed in the presence of either Mn2+ or Mg2+. Manganese ions activated Pol ι in both cases, though to a different extent. In the presence of Mn2+ the Pol ι activity in the basal-cell carcinoma exceeded 2.5-fold that in control cells (benign tumors from the same eyelid region). In extracts of melanoma cells in the presence of either cation, the level of the enzyme activity was approximately equal to that in extracts of cells of surrounding tumor-free tissues as well as in eyes removed after traumas. The distinctive feature of tissue malignancy (in basal-cell carcinoma and in melanoma) was the change in DNA synthesis revealed as Mn2+-activated continuation of DNA synthesis after incorrect incorporation of dG opposite dT in the template by Pol ι. Among cell extracts of different normal mouse organs, only those of testicles exhibited a similar feature. This similarity can be explained by cell division blocking that occurs in all normal cells except in testicles and in malignant cells.

DNA polymerase iota-like activity in crude cell extracts of different mouse organs.

The recently discovered DNA polymerase iota differs greatly from the numerous eukaryotic and prokaryotic DNA polymerases known previously in its ability to catalyze error-prone DNA synthesis. Using homogeneous preparations of the enzyme, it was shown previously that DNA polymerase iota incorporated preferentially dGMP opposite the thymidine of the template in the growing DNA chain. To elucidate the role of this enzyme in the mammals, its activity was assayed in crude cell extracts of different mouse organs. It is shown that the extracts of the brain and testis cells exhibit the highest activity of DNA polymerase iota, which is not in agreement with the results of other authors. The data suggest that the tissue specific expression of DNA polymerase iota is regulated to a significant degree at the posttranscriptional and posttranslational levels.

Trim14 overexpression causes the same transcriptional changes in mouse embryonic stem cells and human HEK293 cells

The trim14 (pub, KIAA0129) gene encodes the TRIM14 protein which is a member of the tripartite motif (TRIM) family. Previously, we revealed high expression levels of trim14 in HIV- or SIV-associated lymphomas and demonstrated the influence of trim14 on mesodermal differentiation of mouse embryonic stem cells (mESC). In the present work, to elucidate the role of trim14 in normal and pathological processes in the cell, we used two different types of cells transfected with trim14: mESC and human HEK293. Using subtractive hybridization and real-time PCR, we found a number of genes which expression was elevated in trim14-transfected mESC: hsp90ab1, prr13, pu.1, tnfrsf13c (baff-r), tnfrsf13b (taci), hlx1, hbp1, junb, and pdgfrb. A further analysis of the trim14-transfected mESC at the initial stage of differentiation (embryoid bodies (EB) formation) showed essential changes in the expression of these upregulated genes. The transfection of trim14 into HEK293 also induced an enhanced expression of the several genes upregulated in trim14-transfected mESC (hsp90ab1, prr13, pu.1, tnfrsf13c (baff-r), tnfrsf13b (taci), and hlx1). Summarizing, we found similar genes that participated in trim14-directed processes both in mESC and in HEK293. These results demonstrate the presence of the similar mechanism of trim14 gene action in different types of mammalian cells.

The expression of HIV‐1 tat and nef genes induces cell‐specific changes in growth properties and morphology of different types of rat cells

Abstract.  Among the viral regulatory genes the tat and nef genes of HIV‐1 encode the proteins playing a central role in viral replication and exerting pleiotropic effects on the survival and growth of the cells. These effects differ in various cell types, possibly due to the use of genes from different HIV‐1 isolates. In this work, we studied the effects of the tat and nef genes on three types of cultured rat cells: primary embryo fibroblasts, pseudonormal Rat‐2, and pheochromocytoma PC12. Both genes affected growth properties and morphology of cells, the effects being cell‐specific. The proliferative activity of both Rat‐2 and PC12 cells was considerably increased after transfection with the tat gene. In primary rat embryo fibroblasts the tat gene induced multilayered foci. More importantly, it was shown that the efficiency of transformation was higher in cells coexpressing tat and nef. The nef gene caused considerable suppression of Rat‐2 cell proliferation, but no changes in their morphology. The nef gene transfection of PC12 cells also led to suppression of their proliferative activity. In addition, cellular agglomerates which were morphologically similar to multinuclear syncytial cells were detected in these cells for the first time.

Evolution of DNA polymerase ι structure and function in eukaryotes

Analysis of DNA polymerase ι (Pol ι) enzymic activity in different classes of eukaryotes has shown that error-prone activity of this enzyme can be found only in mammals, and that it is completely absent from organisms that are at lower stages of development. It was supposed that the emergence of the error-prone Pol ι activity in mammals is caused by structural alteration of the active center. Possible functions of error-prone Pol ι in higher eukaryotes are discussed.

Association between high activity of DNA polymerase iota and the development of human uveal melanoma

Enzymatic activity of DNA polymerase iota (Pol ι) was analyzed in human uveal melanoma cell extracts, using an earlier elaborated approach. The Pol ι activity was observed in seven out of eight malignant tumors, while it was absent in the normal uveal tract cells of the same patients. These findings serve as an additional confirmation of the Pol ι oncogenic potential.