An episomally replicating vector binds to the nuclear matrix protein SAF-A in vivo. (1/28)

pEPI-1, a vector in which a chromosomal scaffold/matrix-attached region (S/MAR) is linked to the simian virus 40 origin of replication, is propagated episomally in CHO cells in the absence of the virally encoded large T-antigen and is stably maintained in the absence of selection pressure. It has been suggested that mitotic stability is provided by a specific interaction of this vector with components of the nuclear matrix. We studied the interactions of pEPI-1 by crosslinking with cis-diamminedichloroplatinum II, after which it is found to copurify with the nuclear matrix. In a south-western analysis, the vector shows exclusive binding to hnRNP-U/SAF-A, a multifunctional scaffold/matrix specific factor. Immunoprecipitation of the crosslinked DNA-protein complex demonstrates that pEPI-1 is bound to this protein in vivo. These data provide the first experimental evidence for the binding of an artificial episome to a nuclear matrix protein in vivo and the basis for understanding the mitotic stability of this novel vector class.  (+info)

Identification of different isoforms of eEF1A in the nuclear fraction of human T-lymphoblastic cancer cell line specifically binding to aptameric cytotoxic GT oligomers. (2/28)

GT oligomers, showing a dose-dependent cytotoxic effect on a variety of human cancer cell lines, but not on normal human lymphocytes, recognize and form complexes with nuclear proteins. By working with human T-lymphoblastic CCRF-CEM cells and by using MS and SouthWestern blotting, we identified eukaryotic elongation factor 1 alpha (eEF1A) as the main nuclear protein that specifically recognizes these oligonucleotides. Western blotting and supershift assays confirmed the nature of this protein and its involvement in forming a cytotoxicity-related complex (CRC). On the contrary, normal human lymphocytes did not show nuclear proteins able to produce CRC in a SouthWestern blot. Comparative bidimensional PAGE and Western-blotting analysis for eEF1A revealed the presence of a specific cluster of spots, focusing at more basic pH, in nuclear extracts of cancer cells but absent in those of normal lymphocytes. Moreover, a bidimensional PAGE SouthWestern blot demonstrated that cytotoxic GT oligomers selectively recognized the more basic eEF1A isoform expressed only in cancer cells. These results suggest the involvement of eEF1A, associated with the nuclear-enriched fraction, in the growth and maintenance of tumour cells, possibly modulated by post-translational processing of the polypeptide chain.  (+info)

MFP1 is a thylakoid-associated, nucleoid-binding protein with a coiled-coil structure. (3/28)

Plastid DNA, like bacterial and mitochondrial DNA, is organized into protein-DNA complexes called nucleoids. Plastid nucleoids are believed to be associated with the inner envelope in developing plastids and the thylakoid membranes in mature chloroplasts, but the mechanism for this re-localization is unknown. Here, we present the further characterization of the coiled-coil DNA-binding protein MFP1 as a protein associated with nucleoids and with the thylakoid membranes in mature chloroplasts. MFP1 is located in plastids in both suspension culture cells and leaves and is attached to the thylakoid membranes with its C-terminal DNA-binding domain oriented towards the stroma. It has a major DNA-binding activity in mature Arabidopsis chloroplasts and binds to all tested chloroplast DNA fragments without detectable sequence specificity. Its expression is tightly correlated with the accumulation of thylakoid membranes. Importantly, it is associated in vivo with nucleoids, suggesting a function for MFP1 at the interface between chloroplast nucleoids and the developing thylakoid membrane system.  (+info)

An interaction between a MYC protein and an EREBP protein is involved in transcriptional regulation of the rice Wx gene. (4/28)

We previously demonstrated that a 31-bp nucleotide sequence located upstream of the rice Wx gene played an important role in its expression. We further showed that this cis-acting regulator interacts with nuclear proteins extracted from developing rice endosperm. We used the 31-bp sequence as bait in a yeast one-hybrid system to isolate several cDNA clones from a rice cDNA expression library. One of these cDNAs encodes a MYC protein, designated OsBP-5, which is 335 amino acids long and contains a putative basic helix-loop-helix-ZIP DNA-binding domain. This domain exhibits 50% amino acid sequence identity with the R/B proteins that regulate the expression of genes involved in anthocyanin biosynthesis in plants. The results of electrophoretic mobility shift assays (EMSAs) and Southwestern gel blots indicate that this protein binds specifically to the CAACGTG motif within the 31-bp sequence. However, by itself, the OsBP-5 protein is unable to trans-activate a lacZ reporter gene controlled by the 31-bp sequence when tested in a yeast expression system. Interestingly, OsBP-5 can trans-activate this reporter gene when another protein, OsEBP-89, a member of the EREBP family of transcription factors, is present. Furthermore, in vitro pull-down experiments show that a protein isolated from developing rice endosperm interacts with the OsBP-5 protein, and Western blots confirm that the interacting protein is OsEBP-89. The formation of a supershift band in EMSAs also indicates that two proteins interact with each other. Interference of OsBP-5 gene expression by double-stranded RNA reduces the amylose content in mature seed of transgenic rice plants but has no visible effect on their phenotype. These results suggest that the OsBP-5 and OsEBP-89 proteins act synergistically, perhaps as a heterodimer, to regulate the transcription of the rice Wx gene.  (+info)

Identification of GAPDH as a protein target of the saframycin antiproliferative agents. (5/28)

Saframycin A (SafA) is a member of a class of natural products with potent antiproliferative effects in leukemia- and tumor-derived cells. This activity is frequently conjectured to derive from the ability of saframycins to covalently modify duplex DNA. We used a DNA-linked affinity purification technique to identify GAPDH as a protein target of DNA-small molecule adducts of several members of the saframycin class. Nuclear translocation of GAPDH occurs upon treatment of cancer cells with saframycins, and depletion of cellular GAPDH levels by small interfering RNA transfection confers drug resistance. Roeder and coworkers have recently suggested that GAPDH is a key transcriptional coactivator necessary for entry into S phase. Our data suggest that GAPDH is also capable of forming a ternary complex with saframycin-related compounds and DNA that induces a toxic response in cells. These studies implicate a previously unknown molecular mechanism of antiproliferative activity and, given that one member of the saframycin class has shown efficacy in cancer treatment, suggest that GAPDH may be a potential target for chemotherapeutic intervention.  (+info)

Borrelia turcica sp. nov., isolated from the hard tick Hyalomma aegyptium in Turkey. (6/28)

Previously, a novel, fast-growing spirochaete was isolated from the hard tick Hyalomma aegyptium, which infests tortoises (Testudo graeca), by using Barbour-Stoenner-Kelly (BSK) II medium; the tick samples were taken from the Istanbul area in northwestern Turkey [Guner et al. (2003). Microbiology 149, 2539-2544]. Here is presented a detailed characterization of the spirochaete. Electron microscopy revealed that strain IST7T is morphologically similar to other spirochaetes of the genus Borrelia and possesses 15 to 16 flagellae that emerge from both polar regions. PFGE analysis revealed the genome to comprise a linear chromosome of approximately 1 Mb; two large linear plasmids of approximately 145 and 140 kb, and several small plasmids ranging from 50 to 20 kb in size were also found. The 16S rRNA gene sequence of this Borrelia isolate exhibited 99.4 to 99.8 % identity with other strains isolated from H. aegyptium and less than 99 % similarity with those of other Borrelia species. A phylogenetic tree, generated from 16S rRNA gene sequences, demonstrated that the spirochaete isolates from H. aegyptium clustered together and branched off from both Lyme-disease-related and relapsing-fever-associated Borrelia species. A single copy of the rrs gene was detected in the genome of strain IST7T by Southern hybridization. DNA-DNA hybridization results showed that strain IST7T was distinct from Lyme-disease-related Borrelia, Borrelia burgdorferi and the relapsing-fever-associated species Borrelia hermsii. The G+C content of strain IST7T is 30.0 mol%. From these genetic features, a novel Borrelia species, Borrelia turcica sp. nov., is proposed; the type strain is IST7T (= JCM 11958T = DSM 16138T).  (+info)

Interaction of an approximately 40 kDa protein from regenerating rat liver with the -148 to -124 region of c-jun complexed with RLjunRP coincides with enhanced c-jun expression in proliferating rat liver. (7/28)

The c-jun belongs to the family of proto-oncogenes and encodes for the protein Jun, a component of transcription factor AP-1 involved in regulation of the expression of genes indispensable for cell proliferation and differentiation. While the role of c-jun in the regulation of such genes has been well examined, the regulation of c-jun in proliferating cells is not fully understood. We have earlier reported that the -148 to -124 region of c-jun is involved in the positive regulation of c-jun transcription, and interacts with a positive regulatory factor (rat liver jun regulatory protein; RLjunRP) present in rat liver. In this investigation, we report that this region is differentially recognized in proliferating liver as evidenced by the formation of a complex, different from that observed with normal liver extract. The new complex appears as early as 2 h after partial hepatectomy and its appearance coincides with the rise in c-jun mRNA levels after partial hepatectomy. In regenerating rat liver nuclear extract, an additional protein of approximately 40 kDa (rRLjunRP) interacts with a pre-existing dimer of RLjunRP complexed with the -148 to -124 region of c-jun to form a slow-migrating complex. rRLjunRP appears to pre-exist in the cytosol and translocate to the nucleus as indicated by the continued presence of the retarded complex in nuclear extract prepared from partially hepatectomized rats treated with cycloheximide. UV crosslinking studies, South-Western blot analysis, SDS/PAGE of affinity-purified factor(s), and 2D-PAGE analysis clearly demonstrate that the additional factor induced in response to growth stimulus is an approximately 40 kDa, that binds with the dimer of RLjunRP and enhances the c-jun transcription.  (+info)

A role for UV-light-induced c-Fos: Stimulation of nucleotide excision repair and protection against sustained JNK activation and apoptosis. (8/28)

UV light (UV-C) is a potent inducer of the c-fos gene. Cells lacking c-Fos are hypersensitive to the cytotoxic effect of UV-C indicating a protective role of c-fos induction. Here we show that cells deficient in c-Fos (fos-/-) are unable to remove cyclobutane pyrimidine dimers (CPDs) from DNA and undergo apoptosis at high frequency via the Fas pathway. We also show that in c-Fos-deficient cells the activation of c-Jun N-terminal kinase (JNK) by UV-C differs from the wild-type (wt, fos+/+). In wt cells JNK activation is transient, returning to control level 5-8 h after treatment, whereas in c-Fos-deficient cells JNK activation was long-lasting and did not return to control level. Inhibition of early JNK activation by the JNK inhibitor SP600125 attenuated CPD repair and increased UV-C induced apoptosis whereas inhibition of late JNK activation attenuated the apoptotic response of c-Fos-deficient cells. Late and sustained activation of JNK resulted in upregulation of fas (CD95, apo-1) ligand and induction of caspase 8-dependent apoptosis. The data indicate that the immediate-early induction of the JNK/c-fos pathway stimulates the repair of UV-C induced DNA lesions that protects against apoptosis. If this does not occur, cells do not recover from transcription blockage leading, as shown for c-Fos-deficient cells, to a reduced expression of MKP1, sustained JNK activation and fasL overexpression that finally results in activation of the death receptor pathway. The data support the hypothesis that non-repaired DNA damage is the cause for the late and sustained activation of the MAP kinase pathway in response to genotoxins.  (+info)