Positive control of transcription initiation in Escherichia coli. A base substitution at the Pribnow box renders ompF expression independent of a positive regulator

Expression of the ompF gene coding for a major outer membrane protein of Escherichia coli is positively regulated by the product of the ompR gene, OmpR. Using an ompF-tet chimera gene, ompF promoter mutants that render the ompF expression independent of the OmpR protein were isolated. In all of the four mutants that were isolated separately, the first base of the Pribnow box was changed from A to T. The mutant promoter did not require the upstream domain of the -35 region that is required for the OmpR-dependent functioning of the wild-type promoter. It is concluded that the domain upstream from the -35 region plays a role in the positive regulation by the OmpR protein. A statistical survey of the E. coli promoter sequence revealed that almost all of the genes that do not require an activator protein for their expression possess T at the first position of the Pribnow box, while the position is occupied by other bases in almost all of the positively regulated genes. Based on these facts, the mechanism of positive regulation of the gene expression by an activator protein is discussed.     

Magnetic susceptibility of hydrogenase from Desulfovibrio vulgaris

Magnetization and magnetic susceptibility measurements revealed that the hydrogenase [EC 1.12.2.1] from Desulfovibrio vulgaris Miyazaki F has an independent unpaired electron in its iron-sulfur cluster. The paramagnetic center of the Desulfovibrio hydrogenase is, therefore, different from that in the Chromatium hydrogenase which interacts with another paramagnetic center, probably nickel.     

Microfibrils in the myotendon junctions.

Myotendon junctions in the rectus abdominis muscles of bull frogs were examined by the fixation combination of tannic acid and glutaraldehyde using electron microscopy. The features observed on myotendon junctions were the following: (1) There were many deep invaginations of muscle cell membrane at the end of the muscle fibers. Terminal thin filaments of myofibrils were attached to the electron-dense layer lining under the muscle cell membrane on the lateral walls of invaginations. (2) The basement membrane covering the muscle cell membrane was thicker in the invaginations than on the other sites of muscle fibers. (3) Collagen fibers in the invaginations gradually tapered off toward the bottom of the invaginations. But it was not seen that the collagen fibers were attached to both the basement membrane and cell membrane of muscle cells. (4) On the observations using the tannic acid-glutaraldehyde fixation, it was clearly seen that the microfibrils extend from the outer leaflets of the cell membrane to the collagen fibers in invaginations via the basement membrane. It was concluded that the myofibrils might be fastened to the collagen fibers of the tendon by the intermediates of the microfibrils.     

Crystallographic data for cytochrome c3 from two strains of Desulfovibrio vulgaris, Miyazaki.

Two crystalline forms of cytochrome c3 isolated from two strains of Desulfovibrio vulgaris, Miyazaki, tentatively designated as D. vulgaris, Miyazki F and D. vulgaris, Miyazaki K, have been found. Both belong to the orthorhombic system, space group P2(1)2(1)2(1), but have different cell dimensions; a=54.1, b=68.9 and c=35.0 A for D. vulgaris, Miyazaki F, and a=43.5, b=41.2, and c=62.9 A for D. vulgaris, Miyazaki K. The asymmetric unit of each crystal contains one molecule of cytochrome c3.     

Glycosphingolipids govern gene expression

To elucidate the biological significance of the lactosylceramide (LacCer) branching in glycosphingolipid (GSL) biosynthesis, we established ganglioside GM3- and lactosylsulfatide SM3-reconstituted cells by introducing the GM3 synthase gene and the sulfotransferase gene, respectively. In SM3-expressing cells, the reduction of beta1 integrin mRNA expression, the reduced adhesivity to fibronectin and laminin, and the suppression of anchorage-independent growth (tumorigenic potential) were observed. On the other hand, in GM3-expressing cells, anchorage-independent growth was promoted and the expression of PDGF alpha receptor mRNA was specifically reduced. Interestingly enough, no change in anchorage-dependent growth was observed in these cells, and tumorigenic signals were controlled selectively in both positive and negative directions. Thus, the spatio-temporal, gene expression control mechanism by individual GSL molecules accumulating in the cell membrane microdomain (raft) has been proven.     

TNFalpha-induced insulin resistance in adipocytes as a membrane microdomain disorder: involvement of ganglioside GM3

Membrane microdomains (lipid rafts) are now recognized as criticalfor proper compartmentalization of insulin signaling, but theirrole in the pathogenesis of insulin resistance has not beeninvestigated. Detergent-resistant membrane microdomains (DRMs),isolated in the low-density fractions, are highly enriched incholesterol, glycosphingolipids and various signaling molecules.Tumor necrosis factor alpha (TNF) induces insulin resistancein type 2 diabetes, but its mechanism of action is not fullyunderstood. In other studies we have found a selective increasein the acidic glycosphingolipid ganglioside GM3 in 3T3-L1 adipocytestreated with TNF, suggesting a specific function for GM3. Inthe DRMs from TNF-treated 3T3-L1 adipocytes, GM3 levels weredoubled compared with results in normal adipocytes. Additionally,insulin receptor (IR) accumulations in the DRMs were diminished,whereas caveolin and flotillin levels were unchanged. Furthermore,insulin-dependent IR internalization and intracellular movementof the IR substrate 1(IRS-1) were both greatly suppressed inthe treated cells, leading to an uncoupling of IR–IRS-1signaling. GM3 depletion was able to counteract the TNF-inducedinhibitions of IR internalization and accumulation into DRMs.Together, these findings provide compelling evidence that ininsulin resistance the insulin metabolic signaling defect canbe attributed to a loss of IRs in the microdomains due to anaccumulation of GM3.     

Partial inhibition of protein synthesis accelerates the synthesis of porphyrin in heme-deficient mutants of Escherichia coli

Mutants of Escherichia coli defective in the HemA protein grow extremely poorly as the result of heme deficiency. A novel hemA mutant was identified whose rate of growth was dramatically enhanced by addition to the medium of low concentrations of translational inhibitors, such as chloramphenicol and tetracycline. This mutant (H110) carries mutation at position 314 in the hemA gene, which resulted in diminished activity of the encoded protein. Restoration of growth of H110 upon addition of the drugs mentioned above was due to activation of the synthesis of porphyrin. However, this activation was not characteristic exclusively of cells with this mutant hemA gene since it was also observed in a heme-deficient strain bearing the wild-type hemA gene. The activation did not depend on the promoter activity of the hemA gene, as indicated by studies with fusion genes. It appears that partial inhibition of protein synthesis via inhibition of peptidyltransferase can promote the synthesis of porphyrin by providing an increased supply of Guamyl-tRNA for porphyrin synthesis. Glutamyl-tRNA is the common substrate for peptidyltransferase and HemA.     

Composition of muscle fibers in the slow loris,using the m. biceps brachii as an example

Histological examination of the skeletal muscle of the slow loris, which displays slow movement and locomotion among the prosimians, revealed a muscle fiber composition which differed from the general condition in mammals. Three types of muscle fiber cells were therefore analyzed quantitatively in order to elucidate their specificity. The skeletal muscle of the limbs of the slow loris was predominantly composed of red muscle fibers (type I) showing persistent tonic contraction.     

Characterization by deletion mutagenesis in vitro of the promoter region of ompF, a positively regulated gene of Escherichia coli

The ompF gene codes for a major outer membrane protein whose expression is positively regulated by the ompR and envZ genes. Two sets of promoter deletions, upstream deletions and downstream deletions, were generated in vitro, and the promoter function was studied by connecting them with the tet genes. One of the hybrid genes thus constructed had a functioning ompF-tet hybrid promoter. The 107 base-pair fragment was found to be functioning as the ompF promoter, 90 nucleotides upstream and 17 nucleotides downstream of the mRNA start site that was also determined in this study. The start site was preceded by a convenient Pribnow box. Although the sequence at the -35 region had a low degree of homology to the consensus sequence, analyses of the hybrid promoter suggested that this region is involved in the promoter function in relation to the Pribnow box. They also indicated that the domain responsible for regulation by the ompR gene is located within the -35 region and its upstream region.