Identification and characterization of rat β-DRE binding factors involved in erythroid-specific induction of transcription

Identifikovali smo i okarakterisali DNK vezujuće proteine koji specifično prepoznaju transkripcioni regulatorni element βDRE iz βbminy-globinskog promotora pacova. βDRE je evolutivno konzervisan u adultnim β-globinskim promotorima sisara. Potvrdili smo da bDRE regulatorni element doprinosi transkripcionoj inducibilnosti βbminy-globinskog gena pacova u pacovskim eritroleukemičnim ćelijama. Korišćenjem "gel mobility shift" i South-Western metoda i kompeticionih eseja, pokazali smo da se za βDRE pacovskog βbminy-globinskog promotora specifično vezuju eritroidno-specifični transkripcioni faktor od 60 kD i dva opšta transkripciona faktora od 35 i 20 kD poreklom iz pacovskih eritroleukemičnih ćelija. Takođe, eritroidna diferencijacija REL ćelija je praćena značajnim povećanjem količine transkripcionog faktora od 60 kD.We have identified and characterized a DNA-binding activity with specificity for the β-globin direct repeat element (βDRE) from rat βbminy-globin promoter, an evolutionarily conserved transcriptional regulatory element in mammalian adult β-globin promoters. We have also confirmed that the βDRE contributed to the transcriptional inducibility of rat βbminy-globin gene in rat erythroleukemia (REL) cells. By using gel mobility shift and South-Western blot competition studies we have shown that 60 kD erythroid-specific transcription factor and 35 kD and 20 kD ubiquitous factors from rat erythroleukemia cells specifically bind to the βDRE from the rat βbminy-globin promoter. Additionally, a significant increase in the quantity of 60 kD transcription factor was observed upon erythroid differentiation of REL cells

Activation of the HSV-TK promoter in control reporter vector pBLCAT5 by liganded nuclear retinoid receptor RXRα

Vektorski sistemi koji sadrže reporterske gene su široko korišćeni za ispitivanje regulatornih DNK elemenata. Promotori patogenih virusa sisara najčešće se koriste kao bazalni promotori u ovim reporterskim sistemima. Opšte je prihvaćena pretpostavka da se pouzdani rezultati mogu dobiti samo pod uslovom da na aktivnost virusnog promotora ne utiču faktori koji deluju in trans ili bilo koji drugi primenjeni eksperimentalni tretmani. U ovom radu smo pokazali da ligandom indukovani nuklearni receptor RXRa aktivira HSV-TK promotor u kontrolnom reporterskom vektoru pBLCAT5. Na osnovu prikazanih rezultata može se zaključiti da vektorske sisteme koji imaju TK kao bazalni promotor treba koristiti samo posle detaljnog testiranja regulacije ekspresije ovog promotora. Pri tome je neophodno primeniti eksperimentalne uslove specifične za pojedina istraživanja da bi se izbegle pogrešne interpretacije rezultata.Widely used reporter vector systems for studying the putative regulatory DNA elements usually contain basal promoters from pathogenic mammalian viruses. It is a common assumption that reliable results can be achieved only if the viral promoter activity is unaffected by transacting factors or any experimental treatment. Here we report that liganded nuclear retinoid receptor RXRa stimulates the HSV-TK promoter in control reporter vector pBLCAT5. Thus, TK driven reporter vectors should be employed only after thorough testing of the regulation of this promoter under experimental stimuli for a particular research purpose in order to avoid unreliable interpretation of the assay results

Trans-activation of the human SOX3 promoter by MAZ in NT2/D1 cells

U ovom radu proučavana je uloga tri visoko konzervisana potencijalna mesta vezivanja za "Myc-associated zinc finger protein" (MAZ) u regulaciji ekspresije humanog SOX3 gena. Eseji izmenjene elektroforetske pokretljivosti u prisustvu antitela na MAZ ukazuju da kompleksi koji se formiraju na dva od tri proučavana mesta u okviru SOX3 promotora sadrže MAZ protein. Takođe, u eksperimentima kotransfekcije smo pokazali da MAZ ima ulogu pozitivnog regulatora transkripcije SOX3 gena, kako u nediferenciranim, tako i u diferenciranim NT2/D1 ćelijama. Iako je MAZ povećao i bazalnu i retinoičnom kiselinom indukovanu promotorsku aktivnost, naši rezultati ukazuju da ovaj transkripcioni faktor ne doprinosi inducibilnosti SOX3 promotora tokom neuralne diferencijacije u prisustvu retinoične kiseline.In this study, we examine the role of three highly conserved putative binding sites for Myc-associated zinc finger protein (MAZ) in regulation of the human SOX3 gene expression. Electrophoretic mobility shift and supershift assays indicate that complexes formed at two out of three MAZ sites of the human SOX3 promoter involve ubiquitously expressed MAZ protein. Furthermore, in cotransfection experiments we demonstrate that MAZ acts as a positive regulator of SOX3 gene transcription in both undifferentiated and RA-differentiated NT2/D1 cells. Although MAZ increased both basal and RA-induced promoter activity, our results suggest that MAZ does not contribute to RA inducibility of the SOX3 promoter during neuronal differentiation of NT2/D1 cells

Up-regulation of the SOX3 gene expression by retinoic acid: characterization of the novel promoter-response element and the retinoid receptors involved

Sox3/SOX3 gene is considered to be one of the earliest neural markers in vertebrates and it is implicated in the genetic cascades that direct brain formation. We have previously shown that early phases of differentiation and neural induction of NT2/D1 embryonal carcinoma cells by retinoic acid ( RA) involve up-regulation of the SOX3 gene expression. Here, we present identification of a novel positive regulatory promoter element involved in RA-dependent activation of the SOX3 gene expression in NT2/D1 cells. This element represents a direct repeat 3-like motif that directly interacts with retinoid X receptor (RXR) alpha in a sequence-specific manner. It is capable of independently mediating the RA effect in a heterologous promoter context and its disruption caused significant reduction of RA/RXR transactivation of the SOX3 promoter. Furthermore, by using synthetic antagonists of retinoid receptors, we have shown for the first time, that RA-induced SOX3 gene expression could be significantly down-regulated by the synthetic antagonist of RXR. Also, this data showed that RXRs, but not RA receptors, are mediators of RA effect on the SOX3 gene up-regulation in NT2/D1 cells. Presented data will be valuable for future investigation of SOX3 gene expression, not only in NT2/D1 model system, but also in diverse developmental, physiological and pathological settings

PCR amplification and sequence analysis of the rat Sox3 gene

Sox3 gen je jedan od markera najranijih faza razvića nervnog sistema kičmenjaka koji je uključen u kontrolu diferencijacije nervnih prekursora. Uprkos činjenici da je genom pacova sekvenciran i javno dostupan, samo parcijalna sekvenca Sox3 gena ove vrste je bila deponovana u bazi podataka. U ovom radu smo primenom PCR-a, sekvenciranja i bioinformatičke analize generisali kompletnu kodirajuću sekvencu Sox3 gena pacova. Analiza dobijene sekvence je pokazala da Sox3 gen kodira protein od 449 amino kiselina. Uporedna analiza ortologih SOX3 proteina pacova i čoveka pokazala je visok stepen evolutivne očuvanosti. Identifikacija i karakterizacija Sox3 gena pacova doprineće boljem razumevanju njegove uloge tokom razvića nervnog sistema i omogućiće bolji uvid u evoluciju ovog gena kod vertebrata.The Sox3 gene is considered to be one of the earliest neural markers in vertebrates, playing a role in specifying neuronal fate. Despite the completion of a rat genome sequencing project, only a partial sequence of the rat Sox3 gene has been available in the public database. Using PCR, sequencing, and bioinformatics tools, in this study we have determined the complete coding sequence of the rat Sox3 gene encoding 449 amino acids. Comparative analysis of rat and human SOX3 proteins revealed a high degree of conservation. Identification of the rat Sox3 gene sequence would help in understanding the biological roles of this gene and provide insight into evolutionary relationships with vertebrate orthologs

Upper Cretaceous geosites on Golija Mountain - objects of geoheritage

The Upper Cretaceous rudist limestones are well-known from several localities in Serbia. Three of these localities (Svilanovo, Bele Vode and Kulizino Selo) are located in SW Serbia, on Golija Mt. These localities are crucial for understanding the development of the Upper Cretaceous shallow-water environments, thus this is an area of great scientific and educational value, particularly considering palaeontology, stratigraphy, palaeoecology and palaeogeography. One of the aims of this paper is to evaluate these geosites and their geotouristic potential, using Geosite Assessment Model (GAM), which is important for their geoconservation as well as for the sustainable development of the area.</p

Direct PCR amplification of the HVSI region in mitochondrial DNA from buccal cell swabs

Amplification of human mitochondrial DNA (mtDNA) has been widely used in population genetics, human evolutionary and molecular anthropology studies. mtDNA hypervariable segments I and II (HVSI and HVSII) were shown to be a suitable tool in genetic analyses due to the unique properties of mtDNA, such as the lack of recombination, maternal mode of inheritance, rapid evolutionary rate and high population-specific polymorphisms. Here we present a rapid and low-cost method for direct PCR amplification of a 330 bp fragment of HVSI from buccal cell samples. Avoiding the DNA isolation step makes this method appropriate for the analysis of a large number of samples in a short period of time. Since the transportation of samples and fieldwork conditions can affect the quality of samples and subsequent DNA analysis, we tested the effects of long-term storage of buccal cell swabs on the suitability of such samples for direct PCR amplification. We efficiently amplified a 330 bp fragment of HVSI even after the long-term storage of buccal cells at room temperature, +4°C or at -20°C, for up to eight months. All examined PCR products were successfully sequenced, regardless of sample storage time and conditions. Our results suggest that the direct PCR amplification of the HVSI region from buccal cells is a method well suited for large-scale mtDNA population studies

SOX transcription factors and glioma stem cells: Choosing between stemness and differentiation

Glioblastoma (GBM) is the most common, most aggressive and deadliest brain tumor. Recently, remarkable progress has been made towards understanding the cellular and molecular biology of gliomas. GBM tumor initiation, progression and relapse as well as resistance to treatments are associated with glioma stem cells (GSCs). GSCs exhibit a high proliferation rate and self-renewal capacity and the ability to differentiate into diverse cell types, generating a range of distinct cell types within the tumor, leading to cellular heterogeneity. GBM tumors may contain different subsets of GSCs, and some of them may adopt a quiescent state that protects them against chemotherapy and radiotherapy. GSCs enriched in recurrent gliomas acquire more aggressive and therapy-resistant properties, making them more malignant, able to rapidly spread. The impact of SOX transcription factors (TFs) on brain tumors has been extensively studied in the last decade. Almost all SOX genes are expressed in GBM, and their expression levels are associated with patient prognosis and survival. Numerous SOX TFs are involved in the maintenance of the stemness of GSCs or play a role in the initiation of GSC differentiation. The fine-tuning of SOX gene expression levels controls the balance between cell stemness and differentiation. Therefore, innovative therapies targeting SOX TFs are emerging as promising tools for combatting GBM. Combatting GBM has been a demanding and challenging goal for decades. The current therapeutic strategies have not yet provided a cure for GBM and have only resulted in a slight improvement in patient survival. Novel approaches will require the fine adjustment of multimodal therapeutic strategies that simultaneously target numerous hallmarks of cancer cells to win the battle against GBM

All-trans retinoic acid influences viability, migration and adhesion of U251 glioblastoma cells

Glioblastoma (GBM) is one of the most aggressive and deadly forms of cancer. Literature data reveals that all-trans retinoic acid (ATRA) has anticancer effects on different types of tumor cells. However, data about the effects of ATRA on glioblastoma cells are contradictory. In this study, we examined whether ATRA treatment affects features of human glioblastoma U251 cells. To that end, the cells were treated with different concentrations of ATRA. Results obtained by MTT and the crystal violet assays imply that ATRA affected the viability of U251 glioblastoma cells in a dose-and time-dependent manner. Fluorescence staining of microtubule cytoskeleton protein a-tubulin revealed that ATRA induced changes in cell morphology. Using semi-quantitative RT-PCR we found that the expression of SOX3 and GFAP genes, as markers of neural differentiation, was not changed upon ATRA treatment. Thus, the observed changes in cell morphology after ATRA treatment are not associated with neural differentiation of U251 glioblastoma cells. The scratch-wound healing assay revealed that ATRA changed the mode of U251 cell migration from collective to single cell motility. The cell-matrix adhesion assay demonstrated that the pharmacologically relevant concentration of ATRA lowered the cell-matrix adhesion capability of U251 cells. In conclusion, our results imply that further studies are needed before ATRA could be considered for the treatment of glioblastoma

Mitochondrial gene pool variability of the residents of the Republic of Serbia

Mitohondrijska DNK (mtDNK) se odlikuje nizom osobina koje je čine pogodnom za istraživanja evolutivne istorije ljudskih populacija koja se zasniva na molekularnim markerima ženske linije nasleđivanja. Tokom poslednje decenije publikovano je više naučnih radova u kojima je analizirana varijabilnost mtDNK u populaciji Srbije primenom markera različite rezolucije uključujući i kompletne genome. U skladu sa očekivanjima zasnovanim na istorijskim, arheološkim i drugim izvorima koji govore u prilog veoma kompleksne istorije populacija na Balkanskom poluostrvu, mtDNK podaci su potvrdili da se srpska populacija odlikuje visokim nivoom raznovrsnosti mtDNK koji je posledica izuzetno složene dinamike ove populacije tokom vremena. Današnji mtDNK profil populacije Srbije ne odstupa od matrilinealnog profila karakterističnog za druge evropske populacije, a genetičke distance pokazuju da ova populacija zauzima centralnu poziciju unutar grupe južnoslovenskih populacija koje se odlikuju visokom heterogenošću. Srpska populacija deli najveći procenat mtDNK haplotipova sa geografski bliskim populacijama Balkanskog poluostrva koje pripadaju južnoslovenskoj grupi, gde su uočeni i potencijalno privatni haplotipovi. Na osnovu filogenetske i filogeografske analize kompletnih mitogenoma u srpskoj populaciji detektovane su retke mtDNK linije, karakteristične za druge regione, poput Bliskog istoka (N1b, HV2), istočne Azije (D4) i Afrike (L2a1), kao i one koje su potencijalno specifične za Balkansko poluostrvo, poput K1a13a1, U4c1b1 i H6a2b. Pored toga, srpska populacija deli određeni broj mtDNK podhaplogrupa sa istočno- i zapadnoslovenskim populacijama kao i sa germanskim populacijama severne i srednje Evrope. Istraživanja varijabilnosti mtDNK su pokazala da se izuzetno velika raznovrsnost mtDNK savremene populacije Srbije može objasniti genetičkim doprinosom kako slovenskih i germanskih, tako i pre-slovenskih populacija koje su naseljavale Balkansko poluostrvo pre Velike seobe naroda.The mitochondrial DNA (mtDNA) is characterized by a number of features that make it suitable for studying the evolutionary history of human populations based on molecular markers with the female-specific line of inheritance. During the last decade, several scientific papers were published in which the mtDNA variability in the population of Serbia was analyzed using markers of different resolution including complete mitogenomes. In accordance with expectations based on historical, archaeological and other sources that speak in favor of a very complex history of populations on the Balkan Peninsula, mtDNA data confirmed that Serbian population is characterized by a high level of mtDNA diversity, which is a consequence of the exceptionally complex dynamics of this population over time. Today’s mtDNA profile of the Serbian population does not differ from the matrilineal landscape characteristic of other European populations, and according to genetic distances, this population occupies a central position within the group of South- Slavic populations characterized by high heterogeneity. The Serbian population shares the highest percentage of mtDNA haplotypes with the geographically close populations of the Balkan Peninsula belonging to the South-Slavic group, where potentially private haplotypes were also observed. Phylogenetic and phylogeographic analysis of complete mitogenomes in the Serbian population revealed rare mtDNA lineages, characteristic of other regions, such as the Middle East (N1b, HV2), East Asia (D4) and Africa (L2a1), as well as those that are potentially specific for Balkan Peninsula, like K1a13a1, U4c1b1 and H6a2b. In addition, Serbian population shares a certain number of mtDNA subhaplogroups with East- and West-Slavic populations as well as with the Germanic populations of Northern and Central Europe. Studies of mtDNA variability have shown that the exceptionally high mtDNA diversity in contemporary Serbian population may be associated with the genetic contribution of both Slavic and Germanic, as well as pre- Slavic populations that inhabited the Balkan Peninsula before the Great Migration