Human TAF-Iα promotes oncogenic transformation via enhancement of cell proliferation and suppression of apoptosis.

Template activating factor-I (TAF-I) is a multifunctional protein involved in various biological processes including the inhibition of histone acetylation, DNA replication, cell cycle regulation, and oncogenesis. Two main TAF-I isoforms with different N-termini, TAF-Iα and TAF-Iß (SET), are expressed in cells. There are numerous data about functional properties of TAF-Iß, whereas the effects of TAF-Iα remain largely unexplored. Here, we employed focus formation and cell proliferation assays, TUNEL staining, cytological analysis, and RT-qPCR to compare the effects of human TAF-Iα and TAF-Iß genes, transiently expressed in Rat2 cells and in Misgurnus fossilis loaches. We found that both TAF-I isoforms possessed equal oncogenic potential in these systems. Furthermore, an overexpression of human TAF-Iα and TAF-Iß in Rat2 cells promoted their proliferation. Accordingly, the mitotic index was increased in the transgenic loaches expressing human TAF-Iα or TAF-Iß. TUNEL assay as well as downregulation of p53 gene and upregulation of bcl-2 gene in these transgenic loaches demonstrated that both isoforms suppressed apoptosis. Thus, TAF-Iα isoform exerts the same oncogenic potential as TAF-Iß, likely by suppressing the apoptosis and promoting cell proliferation.

Expression of the human TRIM14 and its mutant form (P207L) promotes apoptosis in transgenic loaches.

The tripartite-motif (TRIM)14 protein, one of the TRIM family members, was shown to participate in the antiviral and antibacterial defence. Besides, it appears to play an essential role in the processes of oncogenesis. In some types of human tumour cells, TRIM14 has been shown to inhibit apoptosis, while in others-the overexpression of TRIM14 promotes apoptosis. However, whether TRIM14 mediates apoptosis in the normal cells remains unknown. In the present study, we investigated the possible participation of the human TRIM14 gene and its mutant form (620C > T) in the induction of apoptosis in the transgenic larvae loach Misgurnus fossilis L. We observed that the expression of both forms of TRIM14 gene was accompanied by the increase of the frequency of pyknotic nuclei in fish embryos compared to control groups. Accordingly, using the TUNEL assay, the enhanced apoptosis was revealed upon expression of both forms of TRIM14 gene. The transcription of proapoptotic genes (bax, tp53, and casp9) was significantly increased in transgenic loaches expressing human wild-type TRIM14, but remained unchanged upon expression of its mutant form. In addition, the transcription of c-myc was upregulated in transgenic loaches expressing both forms. Thus, it can be assumed that during embryonic development TRIM14 has a proapoptotic effect on the cells via the activation of c-myc, tp53, and bax genes. Apparently, the mutant TRIM14 directs apoptosis via c-myc by p53-independent mechanism.

The ancient small mobilizable plasmid pALWED1.8 harboring a new variant of the non-cassette streptomycin/spectinomycin resistance gene aadA27.

The small mobilizable plasmid pALWED1.8 containing a novel variant of the streptomycin/spectinomycin resistance gene aadA27 was isolated from the permafrost strains of Acinetobacter lwoffii. The 4135bp plasmid carries mobА and mobC genes that mediate its mobilization by conjugative plasmids. The nucleotide sequences of mobА and mobC are similar to those of mobilization genes of the modern plasmid pRAY* and its variants, which contain aadB gene, and are widespread among the pathogenic strains of Acinetobacter baumannii. Almost identical pALWED1.8 variants were detected in modern environmental Аcinetobacter strains. A highly similar plasmid was revealed in a strain of Acinetobacter parvus isolated from mouse intestine. Furthermore, we discovered six previously unidentified variants of plasmids related to pALWED1.8 and pRAY* in public databases. In contrast to most known variants of aadA which are cassette genes associated with integrons, the aadA27 variant harbored by pALWED1.8 is a non-cassette, autonomously transcribed gene. Non-cassette aadA genes with 96% sequence identity to aadA27 were detected in the chromosomes of Acinetobacter gyllenbergii and several uncharacterized strains of Аcinetobacter sp. Moreover, we revealed that the autonomous aadA-like genes are present in the chromosomes of many gram-positive and gram-negative bacteria. The phylogenetic analysis of amino acid sequences of all identified AadA proteins showed the following: (i) cassette aadA genes form a separate monophyletic group and mainly reside on plasmids and (ii) chromosomal non-cassette aadA genes are extremely diverse and can be inherited both vertical and via horizontal gene transfer.

Genetic structure and biological properties of the first ancient multiresistance plasmid pKLH80 isolated from a permafrost bacterium.

A novel multidrug-resistance plasmid, pKLH80, previously isolated from Psychrobacter maritimus MR29-12 found in ancient permafrost, was completely sequenced and analysed. In our previous studies, we focused on the pKLH80 plasmid region containing streptomycin and tetracycline resistance genes, and their mobilization with an upstream-located ISPpy1 insertion sequence (IS) element. Here, we present the complete sequence of pKLH80 and analysis of its backbone genetic structure, including previously unknown features of the plasmid's accessory region, notably a novel variant of the ß-lactamase gene blaRTG-6. Plasmid pKLH80 was found to be a circular 14 835 bp molecule that has an overall G+C content of 40.3 mol% and encodes 20 putative ORFs. There are two distinctive functional modules within the plasmid backbone sequence: (i) the replication module consisting of repB and the oriV region; and (ii) the mobilization module consisting of mobA, mobC and oriT. All of the aforementioned genes share sequence identities with corresponding genes of different species of Psychrobacter. The plasmid accessory region contains antibiotic resistance genes and IS elements (ISPsma1 of the IS982 family, and ISPpy1 and ISAba14 of the IS3 family) found in environmental and clinical bacterial strains of different taxa. We revealed that the sequences flanking blaRTG-6 and closely related genes from clinical bacteria are nearly identical. This fact suggests that blaRTG-6 from the environmental strain of Psychrobacter is a progenitor of blaRTG genes of clinical bacteria. We also showed that pKLH80 can replicate in different strains of Acinetobacter and Psychrobacter genera. The roles of IS elements in the horizontal transfer of antibiotic resistance genes are examined and discussed.

Genomic characterization and integrative properties of phiSMA6 and phiSMA7, two novel filamentous bacteriophages of Stenotrophomonas maltophilia.

Two novel filamentous phages, phiSMA6 and phiSMA7, were isolated from Stenotrophomonas maltophilia environmental strain Khak84. We identified and annotated 11 potential open reading frames in each phage. While the overall layout of the functional gene groups of both phages was similar to that of the known filamentous phages, they differed from them in their molecular structure. The genome of phiSMA6 is a mosaic that evolved by acquiring genes from at least three different filamentous S. maltophilia phages and one Xanthomonas campestris phage related to Cf1. In the phiSMA6 genome, a gene similar to the bacterial gene encoding the mating pair formation protein trbP was also found. We showed that phiSMA6 possesses lysogenic properties and upon induction produces high-titer lysates. The genome of phiSMA7 possesses a unique structure and was found to be closely related to a prophage present in the chromosome of the completely sequenced S. maltophilia clinical strain D457. We suggest that the other three filamentous phages of S. maltophilia described previously also have the capacity to integrate into the genome of their bacterial host.

A new subgroup of the IS3 family and properties of its representative member ISPpy1.

Recently, we described a novel insertion element, ISPpy1, isolated from a permafrost strain of Psychrobacter maritimus. In this work, we demonstrated that ISPpy1 is a member of a novel subgroup of the IS3 family of insertion sequences (ISs) that was not identified and characterized previously. IS elements of this subgroup termed the ISPpy1 subgroup are broadly distributed among different taxa of Eubacteria, including Geobacteraceae, Chlorobiaceae, Desulfobacteraceae, Methylobacteriaceae, Nitrosomonadaceae and Cyanobacteria. While displaying characteristic features of the IS3-family elements, ISPpy1 subgroup elements exhibit some unusual features. In particular, most of them have longer terminal repeats with unconventional ends and frameshifting box with an atypical organization, and, unlike many other IS3-family elements, do not exhibit any distinct IS specificity. We studied the transposition and mutagenic properties of a representative member of this subgroup, ISPpy1 and showed that in contrast to the original P. maritimus host, in a heterologous host, Escherichia coli K-12, it is able to translocate with extremely high efficiency into the chromosome, either by itself or as a part of a composite transposon containing two ISPpy1 copies. The majority of transposants carry multiple chromosomal copies (up to 12) of ISPpy1. It was discovered that ISPpy1 is characterized by a marked mutagenic activity in E. coli: its chromosomal insertions generate various types of mutations, including auxotrophic, pleiotropic and rifampicin-resistance mutations. The distribution of IS elements of the novel subgroup among different bacteria, their role in the formation of composite transposons and the horizontal transfer of genes are examined and discussed.