Sequence and structure of the nucleolin promoter in rodents: characterization of a strikingly conserved CpG island

We report the isolation of the complete genes encoding nucleolin from rat and hamster. The DNA clones were obtained from partial genomic libraries by probing with a genomic DNA fragment containing the leader and promoter regions of the mouse nucleolin gene. We have determined the complete nucleotide sequence of the 5'-terminal region for the three rodent species. The sequenced regions extend over 1 kb downstream and upstream from the cap sites and include a conserved CpG island 1500 nucleotides (nt) long. The 5' end of the CpG island in each species has maintained a long alternating purine-pyrimidine sequence which could adopt a Z-DNA conformation. By sequence comparison, 42 blocks of homology are defined in the 5'-terminal region, of which 36 appear in the CpG island and contain numerous conserved CpG dinucleotides. Two blocks, 110 and 49 nt long, encompassing the cap sites and the region immediately upstream, respectively, present features characteristic of regulated genes: a possible TATA box (ATTA), two pyrimidine-rich nucleotide stretches and two inverted juxtaposed CCAAT-like boxes (GGTTGG). Furthermore, the adjacent upstream conserved region presents features characteristic of housekeeping genes: four G/C boxes, embedded in a high G + C-content sequence, among them one presenting a perfect consensus Sp 1-binding site (GCCCCGCCCC). Among unusual features, we report numerous large G + C-rich conserved sequences located in the first intron. One of these sequences contains two G/C boxes which border a sequence presenting a dyad symmetry (GCGCACGTGCTC). Our findings shed some light on the putative role of the CpG island. We show that CpG-rich sequence motifs are under strong selective pressure over the whole 5'-terminal region and are presumably involved in regulatory mechanisms.     

A radioautographic study of [3H]-A 23187 (calcimycin) in components of the brain. Distribution in different cell types of the diencephalo-mesencephalic roof.

The distribution of the ionophore [3H]-A 23187 was examined by means of light and electron microscopy in elements of the central nervous system located in the diencephalo-mesencephalic roof. A 23187 is not evenly distributed in the components studied (ependyma, secretory ependyma of the subcommissural organ and neurons of the mesencephalon). At the cellular level, A 23187 appears preferentially associated with the cytoplasmic membrane as well as with the internal membranous system.     

Transforming DNA Uptake Gene Orthologs Do Not Mediate Spontaneous Plasmid Transformation in Escherichia coli

Spontaneous plasmid transformation of Escherichia coli occurs on nutrient-containing agar plates. E. coli has also been reported to use double-stranded DNA (dsDNA) as a carbon source. The mechanism(s) of entry of exogenous dsDNA that allows plasmid establishment or the use of DNA as a nutrient remain(s) unknown. To further characterize plasmid transformation, we first documented the stimulation of transformation by agar and agarose. We provide evidence that stimulation is not due to agar contributing a supplement of Ca²⁺, Fe²⁺, Mg²⁺, Mn²⁺, or Zn²⁺. Second, we undertook to inactivate the E. coli orthologues of Haemophilus influenzae components of the transformation machine that allows the uptake of single-stranded DNA (ssDNA) from exogenous dsDNA. The putative outer membrane channel protein (HofQ), transformation pseudopilus component (PpdD), and transmembrane pore (YcaI) are not required for plasmid transformation. We conclude that plasmid DNA does not enter E. coli cells as ssDNA. The finding that purified plasmid monomers transform E. coli with single-hit kinetics supports this conclusion; it establishes that a unique monomer molecule is sufficient to give rise to a transformant, which is not consistent with the reconstitution of an intact replicon through annealing of partially overlapping complementary ssDNA, taken up from two independent monomers. We therefore propose that plasmid transformation involves internalization of intact dsDNA molecules. Our data together, with previous reports that HofQ is required for the use of dsDNA as a carbon source, suggest the existence of two routes for DNA entry, at least across the outer membrane of E. coli.The spontaneous transformation of Escherichia coli with plasmid DNA on nutrient-containing agar plates was described in at least three independent articles (14, 23, 24). However, no attempt to characterize the mechanism of plasmid DNA uptake has been reported. Genomic analysis revealed the presence in E. coli of a set of genes homologous to those required for DNA uptake in naturally transformable species, including the gram-positive Bacillus subtilis and Streptococcus pneumoniae and the gram-negative Haemophilus influenzae and Neisseria gonorrhoeae (9). The machine they potentially encode would allow the uptake of single-stranded DNA (ssDNA) from an exogenous double-stranded DNA (dsDNA) substrate in E. coli (Fig. ​(Fig.1).1). HofQ (called ComE in reference 7) is the ortholog of the PilQ secretin of N. gonorrhoeae, which constitutes a transmembrane channel required for exogenous dsDNA to traverse the outer membrane (OM) and reach the so-called transformation pseudopilus (8). According to the Bacillus subtilis paradigm (8), assembly of the pseudopilus requires a prepilin peptidase (PppA; called PilD in reference 7), a traffic NTPase (HofB; called PilB in reference 7), and a polytopic membrane protein (HofC; called PilC in reference 7). The pseudopilus, which would include PpdD (called PilA in reference 7), provides access for dsDNA to its receptor, YbaV (called ComE1 in reference 7), through the peptidoglycan. Degradation of one strand by an unidentified nuclease (N) would allow uptake of ssDNA through YcaI (called Rec2 in reference 7), a channel in the inner membrane. Finally, DprA (also named Smf) would be required to protect internalized ssDNA from endogenous nucleases, as shown in S. pneumoniae (4), and to assist the processing of ssDNA into transformants (16).Open in a separate windowFIG. 1.Diagrammatic representation of the putative E. coli DNA uptake machine. The E. coli orthologues of proteins required involved in the uptake of transforming DNA in naturally transformable species, including B. subtilis, S. pneumoniae, H. influenzae, and N. gonorrhoeae, were identified by genomic analysis (9). GspD is a PilQ paralogue (25% identity over 278 residues), which was considered in the present study as a possible alternative route for dsDNA across the OM. A prepilin peptidase (PppA; called PilD in reference 7) required for maturation and export of proteins constituting the transformation pseudopilus (see Table S1 in the supplemental material) is not drawn on this diagram. (Additional information regarding the relationship between E. coli and H. influenzae transformation genes, and a table listing the various alternative names used in the literature are available in the supplemental material.). Red crosses indicate components of the putative DNA uptake machine inactivated during this work. IM, inner membrane.In H. influenzae, transformation genes are preceded by unusual CRP (for cyclic AMP receptor protein) binding sites, now called CRP-S (7), that absolutely require a second protein, Sxy (also called TfoX), in addition to CRP for induction (19). Interestingly, bioinformatics analysis revealed the conservation of CRP-S sites in front of the corresponding E. coli genes (7), including all of the genes encoding the proteins shown in Fig. ​Fig.11 (except GspD). Furthermore, some of these genes were experimentally demonstrated to require CRP, cAMP (CRP''s allosteric effector), and Sxy for induction in E. coli, providing support to the view that CRP-S sites control a bona fide transformation regulon in this bacterium (7). However, the involvement of E. coli transformation genes in DNA uptake has not been documented, except for hofQ, which was reported to be required for the use of dsDNA as a nutrient (11, 18). Although the functionality of the E. coli transformation genes has not been confirmed experimentally, it is of note that the bioinformatics identification of a complete set of transformation genes in two other species not previously known to be naturally transformable, Streptococcus thermophilus and Bacillus cereus, opened the way to the demonstration of genetic transformation in these species (6, 15a).To characterize further spontaneous plasmid transformation in E. coli, we first identified parameters affecting plasmid transformation frequencies on plates. We then undertook to inactivate genes encoding the putative transformation-related DNA uptake machinery of E. coli (Fig. ​(Fig.1)1) and to compare the rate of spontaneous plasmid transformation in the corresponding mutants and in their wild-type parent. In addition, to get an insight into the process of plasmid DNA entry, we characterized the kinetics of plasmid monomer transformation because it was shown in S. pneumoniae that regeneration of an intact plasmid replicon requires the independent uptake (via the transformation machine) of complementary ssDNA from two monomers (21). Finally, we discuss the possible significance of our data regarding the entry of exogenous dsDNA in E. coli in the light of previous findings on the use of dsDNA as a carbon source in this species (11, 18).     

The global nutritional regulator CodY is an essential protein in the human pathogen Streptococcus pneumoniae

CodY is a global regulator highly conserved in low-G+C Gram-positive bacteria. It plays a key role in the adaptation of Bacillus subtilis to nutritional limitation through repression of a large gene set during exponential growth and relief of repression upon starvation. In several pathogenic bacteria, CodY regulates major virulence genes. Our interest in Streptococcus pneumoniae CodY originates from our observations that the oligopeptide permease Ami was involved in repression of competence for genetic transformation. We hypothesized that peptide uptake through Ami feeds amino acid pools, which are sensed by CodY to repress competence. As our initial attempts at inactivating codY failed, we launched an in-depth analysis into the question of the essentiality of codY. We report that codY cannot be inactivated unless a complementing ectopic copy is present. We obtained genetic evidence that a recently published D39 codY knock-out contains additional mutations allowing survival of codY mutant cells. Whole genome sequencing revealed mutations in fatC, which encodes a ferric iron permease, and amiC. This combination of mutations was confirmed to allow tolerance of codY inactivation. The amiC mutation is in itself sufficient to account for the strong derepression of competence development observed in D39 codY cells.     

<Emphasis Type="Italic">In vitro</Emphasis> and <Emphasis Type="Italic">ex vivo</Emphasis> effect of hyaluronic acid on erythrocyte flow properties

Background  

Hyaluronic acid (HA) is present in many tissues; its presence in serum may be related to certain inflammatory conditions, tissue damage, sepsis, liver malfunction and some malignancies. In the present work, our goal was to investigate the significance of hyaluronic acid effect on erythrocyte flow properties. Therefore we performed in vitro experiments incubating red blood cells (RBCs) with several HA concentrations. Afterwards, in order to corroborate the pathophysiological significance of the results obtained, we replicated the in vitro experiment with ex vivo RBCs from diagnosed rheumatoid arthritis (RA) patients, a serum HA-increasing pathology.     

Semi-synthesis, topoisomerase I and kinases inhibitory properties, and antiproliferative activities of new rebeccamycin derivatives

In the course of structure-activity relationship studies, new rebeccamycin derivatives substituted in 3,9-positions on the indolocarbazole framework, and a 2',3'-anhydro derivative were prepared by semi-synthesis from rebeccamycin. The antiproliferative activities against nine tumor cell lines were determined and the effect on the cell cycle of murine leukemia L1210 cells was examined. Their DNA binding properties and inhibitory properties toward topoisomerase I and three kinases PKCzeta, CDK1/cyclin B, CDK5/p25 and a phosphatase cdc25A were evaluated. The 3,9-dihydroxy derivative is the most efficient compound of this series toward CDK1/cyclin B and CDK5/p25. It is also characterized as a DNA binding topoisomerase I poison. Its broad spectrum of molecular activities likely accounts for its cytotoxic potential. This compound which displays a tumor cell line-selectivity may represent a new lead for subsequent drug design in this series of glycosylated indolocarbazoles.