The role of microRNAs in endometrial cancer and influence on future therapy: focusing on miRNA-21.

MicroRNAs are small noncoding polynucleotides, which are involved in numerous biological processes including cell proliferation, differentiation, embryonic development, as well as regulation of cell death and survival. Recent investigations have shown impact of microRNAs on cancers prognosis and diagnosis. Current review focused on the role of microRNA-21 in cancers tumorigenesis. Endometrial cancer is the most common gynecological malignancy and the fourth most common in general classification of cancers in Western Europe; thus discovering new molecules may become a useful diagnostic tool. Furthermore, in this review, the authors emphasized microRNAs having considerable influence on endometrial cancer development. Finally, they highlighted the role of microRNAs as a target for future therapy and circulating microRNAs as a potential biomarker in malignancies.

Efficient transcriptional antitermination from the Escherichia coli cytoplasmic membrane.

The BglG protein is a transcriptional antiterminator acting within the beta-glucoside operon of Escherichia coli by binding to a specific sequence motif in the growing mRNA. Binding of BglG prevents formation of the terminator stem-loop structure, thereby causing the RNA polymerase to continue transcription. Activity of BglG is modulated in a complex way by antagonistically acting phosphorylations in response to the availability of beta-glucosidic substrates and to the catabolic state of the cell. The enzymes responsible for these phosphorylations are members of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) that represents a central carbohydrate uptake and signal transduction system. As these enzymes are believed to all form higher-order complexes associated with the cytoplasmic membrane, we tested whether or not BglG would remain active when artificially anchored to its presumptive site of regulation, the inner membrane. We show that the membrane-anchored protein indeed efficiently catalyzes transcriptional antitermination. Moreover, the membrane-attached BglG remains regulated by the PTS. Thus, a membrane-bound regulatory RNA binding protein can potentially interact fast enough with its target within the nascent transcript and cause the transcriptional machinery to proceed, before transcriptional termination would occur. Consequently, there is no principal necessity for an RNA-binding transcriptional regulator like BglG to leave the inner membrane, a potential regulatory site, and migrate to the site of transcription, the nucleoid.

Genetic organization of insertion element IS2 based on a revised nucleotide sequence.

We identified a transposable element resident in the chromosome of Escherichia coli K-12 strain HB101. This is an approx. 4400-bp-long transposon flanked by two copies of insertion sequence (IS) 1 element in direct orientation. One of the IS1 elements was found to be integrated into an IS2 element between IS2 bp 139 and bp 140 with the large moiety of IS2 within the transposon. The sequence of this part of IS2 differs from the published sequence of galOP-308::IS2 at a number of positions. Restriction analysis of the published allele, however, indicated that both alleles may in fact be identical. Since six of the eight differences found alter open reading frames, the revised sequence presents a new outlook for the potential genetic organization of IS2.

Two site-specific endonucleases BinSI and BinSII from Bifidobacterium infantis.

Two site-specific endonucleases, BinSI and BinSII, were isolated from Bifidobacterium infantis S76e. BinSI was found to be an isoschizomer of EcoRII, while BinSII was shown to have the same sequence and cutting specificity as BbeI, 5'-GGCGC decreases C-3'. Both BinSII- and BbeI-generated DNA fragments could be ligated with HaeII-generated DNA fragments.

Bacterial proteins with N-terminal leader sequences resembling mitochondrial targeting sequences of eukaryotes.

Amphipathic, alpha-helical, leader sequences, analogous to those that direct nuclear-encoded eukaryotic proteins into mitochondria, have been found in one and only one class of bacterial integral membrane proteins. These bacterial proteins are the sugar permeases of the phosphoenolpyruvate-dependent phosphotransferase system. The amphipathic leader sequence in each of these proteins is terminated by a helix breaker, either a prolyl residue or 2 adjacent glycyl residues. Preliminary evidence suggests that these leader sequences function to target the proteins to the envelope fraction of the prokaryotic cell during their biosynthesis.

Glucocorticoid effects on axonal regeneration of retinal ganglion cells in vitro.

Although glucocorticoids are commonly used for the treatment of optic nerve inflammations, little is known about direct effects of steroids on retinal nerve cells. In this study, the neuritogenic effect of prednisolone on retinal ganglion cells was investigated using retinal organ cultures derived from adult rats and embryonic chickens. The numbers of axons growing from retinal ganglion cells (RGC) in vitro were counted to measure the regenerative propensity at various corticosteroid concentrations. Prednisolone exerted a dose-dependent neuritogenic influence on RGC of either origin. The effect was highest at a concentration of 25 µg/ml culture medium, where axon numbers increased to 237% (rat) and 166% (chick) of the control values. This was highly significant (t-test: P < 0.01 and P < 0.005, respectively). High corticosteroid concentrations (>125 µg/ml) produced toxic effects, as revealed by low axon numbers as well as morphological alterations of the explants.

Gal mRNA initiated within IS2.

A strain of E. coli carrying IS2 in the control region of the gal operon has been found to revert to a constitutive phenotype. These revertants can be divided into two classes, which differ in their rate of enzyme synthesis and gal transcription. One revertant synthesizes the gal messenger at a low level (low level revertants), the other at a higher level that exceeds that of the induced wild type two- to three-fold (high level revertants). In both cases it has been shown that the gal messenger is covalently bound to IS2-RNA, which is transcribed from IS2 in the original orientation. The evidence suggests, that the section of the IS2-element, from which this RNA is transcribed in the case of the high level revertants is smaller than 50 nucleotides long: i.e. the resolution of the hybridization method used for the detection of sequence homologies exceeds that of the electron microscopical heteroduplex technique.

A control system which causes alternating turn-off/turn-on of transcription on insertion element IS1.

Presence of insertion element IS1 within an operon leads to absence of expression of genes distal to its integration site. This strong polar effect is observed irrespective of the orientation of IS1. Here I report on a mutant of E. coli K12 which allows turn-on of genes located distally to IS1. This turn-on is due to activation of transcription starting within IS1. The activation of transcription is under the control of a regulatory locus (sis1) which acts in trans. The regulatory locus itself changes its state, which leads to an alternating turn-off/turn-on of genes located distal to IS1: The rate of turn-off was observed with a frequency of about 0.3% (per cell and per generation), that of turn-on with a frequency of about 0.015% (per cell and per generation).

Regulation of the bgl operon of Escherichia coli by transcriptional antitermination.

The bgl operon of Escherichia coli encodes all functions necessary for the regulated uptake and utilization of aryl beta-glucosides. The operon is unusual, however, in that it is cryptic in wild-type strains, requiring activation by mutational events. The vast majority of these mutations are due to transposition of insertion elements into the promoter region of the operon. In this report we show that integration of IS5 into the vicinity of the bgl promoter (P0) enhances its activity by greater than 60-fold thereby activating the operon. In the activated state the operon is subject to induction by substrate. Recent studies have shown that induction of the bgl operon by substrate involves antitermination within the leader of the operon. We now show that substrate-dependent regulation involves specific termination/antitermination of transcription at two signal structures flanking the first gene of the operon, bglG. Antitermination is mediated by the product of gene bglG. In the absence of substrate this antitermination is prevented by the action of the product of gene bglF (the second gene of the operon), which encodes the beta-glucoside-specific transport protein (enzymeIIBgl of the phosphoenolpyruvate-dependent phosphotransferase system, PTS) resulting in repression of the operon. The bgl promoter (P0) is not subject to substrate-dependent regulation. The bgl operon has two additional promoters (P1 and P2) located within the terminators, which could also participate in regulation.

Catabolite control of Escherichia coli regulatory protein BglG activity by antagonistically acting phosphorylations.

In bacteria various sugars are taken up and concomitantly phosphorylated by sugar-specific enzymes II (EII) of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The phosphoryl groups are donated by the phosphocarrier protein HPr. BglG, the positively acting regulatory protein of the Escherichia coli bgl (beta-glucoside utilization) operon, is known to be negatively regulated by reversible phosphorylation catalyzed by the membrane spanning beta-glucoside-specific EIIBgl. Here we present evidence that in addition BglG must be phosphorylated by HPr at a distinct site to gain activity. Our data suggest that this second, shortcut route of phosphorylation is used to monitor the state of the various PTS sugar availabilities in order to hierarchically tune expression of the bgl operon in a physiologically meaningful way. Thus, the PTS may represent a highly integrated signal transduction network in carbon catabolite control.

Beta-glucoside permease represses the bgl operon of Escherichia coli by phosphorylation of the antiterminator protein and also interacts with glucose-specific enzyme III, the key element in catabolite control.

The beta-glucoside (bgl) operon of Escherichia coli is subject to both positive control by transcriptional termination/antitermination and negative control by the beta-glucoside-specific transport protein, an integral membrane protein known as enzyme IIBgl. Previous results led us to speculate that enzyme IIBgl exerts its negative control by phosphorylating and thereby inactivating the antiterminator protein, BglG. Specifically, our model postulated that the transport protein enzyme IIBgl exhibits protein-phosphotransferase activity in the absence of beta-glucosides. We now present biochemical evidence that the phosphorylation of protein BglG does indeed occur in vivo and that it is accompanied by the loss of antitermination activity. BglG persists in the phosphorylated state in the absence of beta-glucosides but is rapidly dephosphorylated when beta-glucosides become available for transport. Our data also suggested specific interactions between the beta-glucoside transport protein and the glucose-specific enzyme III (enzyme IIIGlc), a component of glucose transport and a key element in regulation of catabolite repression. These observations indicate that enzyme IIIGlc may, in conjunction with enzyme IIBgl, modulate the transport of beta-glucosides and the phosphorylation of the antiterminator protein. In the absence of both sugars, when the catabolite-controlled promoter of the operon is derepressed, enzyme IIIGlc may mediate tight repression of antitermination.

IS5: a mobile enhancer of transcription in Escherichia coli.

The cryptic bgl operon of Escherichia coli is activated by the spontaneous insertion of mobile DNA elements. Screening of a collection of such mutations revealed insertion of the 1195-base-pair element IS5 into various positions both upstream and downstream of the bgl promoter P0. Activation of the operon was in all cases attributable to enhancement of P0 activity. Introduction of internal deletions into IS5 almost completely abolished P0 enhancement, demonstrating that enhancement is not simply the result of mutational inactivation of some inhibitory sequences. Intact copies of IS5 in trans restored the enhancing activity of the deletion derivatives. The trans-activator is encoded by IS5 gene ins5A, an essential transposition function. Activation of gene expression by means of interaction of a defective mobile element in cis with functions encoded by a nondefective element in trans has so far been described only for a maize controlling element.

Second-element turn-on of gene expression in an IS1 insertion mutant.

To learn more about the ways in which genes silenced by insertion mutations can be reactivated, we have undertaken a systematic investigation of Gal+ revertants of the polar mutant galOP-306::IS1 in Escherichia coli K12. The selective conditions used excluded reversion to wild type by precise excision of IS1. In this system (which resided on a multi-copy plasmid) reversion to the Gal+ phenotype occurred with a frequency of about 10(-7) per cell and per generation. Analysis of the revertants revealed that - with the single exception of the previously published chromosomal mutant sis1 - alterations in the structure of IS1 lead to reactivation of gal operon expression. These events fall into four classes: (I) insertion of IS2 at position 327 in IS1, insertion of IS2 at position 687 in IS1, (III) insertion of a hitherto undetected mobile element, IS150, at position 387, (IV) a 16-bp deletion encompassing IS1 coordinates 553-568. Of some 200 independent reversion events studied, all but one were of types I-III i.e. they involved the intervention of a second mobile element.

IS150: distribution, nucleotide sequence and phylogenetic relationships of a new E. coli insertion element.

Recently we identified the new insertion (IS) sequence IS150 in various strains of Escherichia coli K-12. We have screened other strains of E. coli and Salmonella typhimurium for the presence of homologous sequences. The strains of E. coli K-12 and W tested contain one or more copies of homology to IS150. We have also determined the complete nucleotide sequence of a copy of IS150 inserted into IS1. Comparison of nucleotide and deduced amino acid sequences of IS150, IS2, IS3, IS51, IS600 and IS629 reveals significant homologies suggesting that these elements are members of a family of phylogenetically related insertion sequences.

Beta-glucoside (bgl) operon of Escherichia coli K-12: nucleotide sequence, genetic organization, and possible evolutionary relationship to regulatory components of two Bacillus subtilis genes.

Wild-type Escherichia coli cells are unable to grow on beta-glucosides. Spontaneous mutants arise, however, which are able to utilize certain aromatic beta-glucosides such as salicin or arbutin as carbon sources, revealing the presence of a cryptic operon called bgl. Mutations activating the operon map within (or close to) the promoter region of the operon and are due to the transposition of an IS1 or IS5 insertion element into this region. This operon was reported to consist of three genes coding for a phospho-beta-glucosidase, a specific transport protein (enzyme IIBgl), and a positively regulating protein. We have defined the extent and location of three structural genes, bglC, bglS, and bglB, and have determined their DNA sequence. The amino acid sequences deduced from the open reading frames together with deletion and subcloning analyses suggest that the first gene, bglC, codes for the regulatory protein, the second, bglS, codes for the transport protein, and the third, bglB, for phospho-beta-glucosidase. A fourth gene may exist which codes for a product of unknown function. We discuss structural features of the DNA sequence which may bear on the regulation of the operon. Homologies to sequences preceding the gene for an excreted levansucrase of Bacillus subtilis, which are known to be involved in the regulation of this gene, and to sequences preceding the gene for an excreted beta-endoglucanase of B. subtilis, for which data pertaining to regulation are not yet available, suggest a close evolutionary relationship among the regulatory components of all three systems.

Insertion element IS5 contains a third gene.

We have previously shown that IS5 contains two genes encoded on opposite DNA strands within the same stretch of DNA. Here we present evidence that a third gene and its promoter are present on IS5. The newly discovered gene, ins5C , is contained within the longest gene of IS5, ins5A , but encoded by the complementary DNA strand. The three genes comprise a total of 519 codons present on the 1195-bp element. The arrangement of these genes represents a coding structure of unprecedented compactness.

The gene bglH present in the bgl operon of Escherichia coli, responsible for uptake and fermentation of beta-glucosides encodes for a carbohydrate-specific outer membrane porin.

The cryptic gene bglH from the Escherichia coli chromosome was cloned into a tacOP-driven expression vector. The resulting plasmid was transferred into the porin-deficient E. coli strain KS26 and the protein was expressed by addition of IPTG. The BglH protein was localized in the outer membrane. It was purified to homogeneity using standard methods. Reconstitution experiments with lipid bilayer membranes defined BglH as a channel-forming component, i.e. it is an outer membrane porin. The single-channel conductance of BglH (560 pS in 1 M KCl) was only one-third of that of the general diffusion porins of E. coli outer membrane. The presence of carbohydrates in the aqueous phase led to a dose-dependent block of ion transport through the channel, similar to that found for LamB (maltoporin) of E. coli and Salmonella typhimurium, which means that BglH is a porin specific for the uptake of carbohydrates. The binding constants of a variety of different carbohydrates were calculated from titration experiments of the BglH-induced membrane conductance. The tightest binding was observed with the aromatic beta-D-glucosides arbutin and salicin, and with gentibiose and cellobiose. Binding of maltooligosaccharides to BglH was in contrast to their binding to LamB in that it was much weaker, indicating that the binding site of BglH for carbohydrates is different from that of LamB (maltoporin). The kinetics of cellopentaose binding to BglH was investigated using the carbohydrate-induced current noise and was compared with that of cellopentaose binding to LamB (maltoporin) and ScrY (sucroseporin).

A 3.6-kbp segment from the vir region of Ti plasmids contains genes responsible for border-sequence-directed production of T region circles in E. coli.

The vir region of Ti plasmids is responsible for the transfer of the T region from Agrobacteria to plant cells; previous experiments suggested that formation of independent T region DNA circles is one step in this process. To study this step in Escherichia coli, we developed a binary vector system. One plasmid (= substrate) contains correctly oriented right and left borders from octopine plasmid pTiAch5. A gene with a counterselectable function (galK) was cloned between these borders. The galK gene is under control of the tac promoter-operator and the lac repressor with the laci gene also in the selection cassette. This construction allows determination of substrate plasmid mutants which have lost the selectable galK function. The second component of the system is one of a set of compatible plasmids harbouring various cloned parts from the vir region of nopaline plasmid pTiC58. A 3.6-kbp segment of the vir region turned out to be necessary and sufficient for production of substrate plasmid mutants which represented the equivalent of the T region containing a complete left border. From this vir region fragment four discrete proteins were expressed in minicells. The coding regions were mapped to a part conserved in nopaline and octopine plasmids; in the latter it appears to correspond to virC/D.

LicT, a Bacillus subtilis transcriptional antiterminator protein of the BglG family.

Gene licS of Bacillus subtilis encodes an excreted Beta-1,3-1,4-endoglucanase necessary for lichenan utilization. Upstream of licS we found a gene (termed licT) together with its promoter which encodes a transcriptional antiterminator of the BglG family. Genes licT and licS are separated by a palindromic sequence (lic-t) reminiscent of transcriptional terminators recognized by the antiterminator proteins of the BglG family. The LicT protein can prevent termination at terminator lic-t and also at terminator t2 of the Escherichia coli bgl operon and BglG prevents termination at lic-t. The role of LicT in licS regulation by preventing termination at its terminator lic-t appears to be limited since expression of licS is inducible only two- to threefold. This limited regulation is mainly due to a high basal level of licS expression which can in part be attributed to the presence of a second promoter preceding licS and located downstream of lic-t. However, disruption of gene licT leads not only to loss of inducibility of licS but also to loss of growth on lichenan or on its degradation products, indicating its stringent role in beta-glucan utilization.