Analysis of Cre1 binding sites in the Trichoderma reesei cbh1 upstream region

Abstract A 1.5-kb XbaI-SacII fragment containing the upstream region of the Trichoderma reesei cellobiohydrolase I gene ( cbh1 ) has been sequenced. The 1.5-kb fragment contains eight 6-bp sites having an identical or similar sequence to the consensus sequence for binding a catabolite repressor, Aspergillus nidulans CreA. Results of binding assays with the maltose-binding protein: :Cre1(10–131) fusion protein (Cre1 is a catabolite repressor of T. reesei ) and the cbhI upstream region revealed that a 504-bp XbaI-NspV fragment (nucleotide position − 1496 to − 993) bearing three 6-bp sites, Al, A2, and A3, and a 356-bp NspV-MunI fragment (nucleotide position −994 to −639) bearing three 6-bp sites, B1, B2, and B3, were shifted in the electrophoretic mobility shift assay. DNase I footprinting experiments showed that the 6-bp sites A2, B1, B2, and B3 were protected from DNase I digestion.     

Silicon carbide fiber-mediated DNA delivery into cells of wheat (Triticum acstivum L.) mature embryos

We have demonstrated that foreign DNA can be delivered into cells of mature embryos of wheat (Triticum aestivum L.) using silicon carbide fibers (SCF). The highest transient expression of thegusA (GUS) gene was detected when dry embryos were vortexed for 10–30 min in a SCF-DNA solution containing 90–120 g/l of sucrose. Up to 100 (on average 20–40) blue expression units per embryo were observed. Scutellum side and epiblast of the intact wheat embryos are preferentially transformed. When embryos with the coleoptilar tip removed were treated and allowed to germinate, GUS staining was observed in emerging leaf tissues. The potential of this new approach for stable transformation of wheat is under investigation. It has been found that callus tissues induced from the SCF treated embryos contain GUS-expressing sectors one month after treatment.     

Cloning, sequencing, and expression of thermophilic Bacillus sp. strain TB-90 urease gene complex in Escherichia coli.

The urease of thermophilic Bacillus sp. strain TB-90 is composed of three subunits with molecular masses of 61, 12, and 11 kDa. By using synthetic oligonucleotide probes based on N-terminal amino acid sequences of each subunit, we cloned a 3.2-kb EcoRI fragment of TB-90 genomic DNA. Moreover, we cloned two other DNA fragments by gene walking starting from this fragment. Finally, we reconstructed in vitro a 6.2-kb DNA fragment which expressed catalytically active urease in Escherichia coli by combining these three DNA fragments. Nucleotide sequencing analysis revealed that the urease gene complex consists of nine genes, which were designed ureA, ureB, ureC, ureE, ureF, ureG, ureD, ureH, and ureI in order of arrangement. The structural genes ureA, ureB, and ureC encode the 11-, 12-, and 61-kDa subunits, respectively. The deduced amino acid sequences of UreD, UreE, UreF, and UreG, the gene products of four accessory genes, are homologous to those of the corresponding Ure proteins of Klebsiella aerogenes. UreD, UreF, and UreG were essential for expression of urease activity in E. coli and are suggested to play important roles in the maturation step of the urease in a co- and/or posttranslational manner. On the other hand, UreH and UreI exhibited no significant similarity to the known accessory proteins of other bacteria. However, UreH showed 23% amino acid identity to the Alcaligenes eutrophus HoxN protein, a high-affinity nickel transporter.     

Meristem-specific gene expression directed by the promoter of the S-phase-specific gene,cyc07, in transgenic Arabidopsis

A genomic clone for the cyc07 gene, which is expressed specifically at the S phase during the cell cycle in synchronous cultures of periwinkle (Catharanthus roseus) cells, was isolated. Determination of the nucleotide sequence of the clone revealed that the cyc07 gene consists of seven exons separated by six introns. Genomic Southern analysis indicated that the cyc07 gene is present as a single copy per haploid genome in periwinkle. Expression of related genes was detected in a wide range of other plants. Transgenic Arabidopsis plants were generated that expressed the gene for -glucuronidase (GUS) under the control of the promoter of the cyc07 gene. The tissue-specific pattern of expression directed by the promoter was investigated by analysis of GUS activity. Histochemical tests demonstrated that 589 bp of the 5-upstream sequence of the cyc07 gene could direct specifical expression of the GUS reporter gene in meristematic tissues in transgenic plants. The spatial pattern of expression directed by the promoter was closely correlated with meristematic activity and cell proliferation, suggesting an association between the function of the cyc07 gene and cell proliferation.     

High-resolution separation of disaccharide and oligosaccharide alditols from chondroitin sulphate, dermatan sulphate and hyaluronan using CarboPac PA1 chromatography

Recent literature indicates that specific glycosaminoglycanstructures are involved in various biological processes, suchas anticoagulation, growth factor activation and viral infection.The initial step in the structural analysis of glycosaminoglycansis a definitive compositional analysis of its characteristicdisaccharide repeat structures. Current chromatographic or electrophoreticprocedures may have limitations in analysing glycosaminoglycansamples that are in low abundance, contain novel structuresthat need to be further characterized, or are metabolicallylabelled from radioactive precursors as a result of biosyntheticexperiments. This study presents a new methodology for analysingdisaccharides and oligosaccharides derived from chondroitinsulphate, dermatan sulphate and hyaluronan that fulfils theabove criteria. The procedure involves the separation of reducedforms of these glycoconjugates on a CarboPac PA1 column usingalkaline eluants. This study adopted a strategy which uses specificenzymes to release these disaccharides from their glycosaminoglycanforms. A borohydride reduction reaction was modified to be compatiblewith the buffer conditions commonly used with these enzymesin order to quantitatively reduce the disaccharides to theiralditol forms (thereby stabilizing them to alkaline pH). Chromatographyconditions were established which separated all known disaccharidealditol structures from chondroitin sulphate, dermatan sulphateand hyaluronan with extremely high resolution in a single run.Integrated pulsed amperometry was compared to UV absorbancemeasurement at 232 nm as two sensitive methods for detectingthese reduced disaccharides; most of them could be routinelydetected in the range of 50–500 ng. Data are presentedapplying this method to quantify hyaluronan in a biologicalsample which contains {small tilde}5000 cells and only {smalltilde}10 ng of hyaluronan. Additional data are presented todemonstrate that this procedure will also separate oligosaccharidealditols derived from hyaluronan. borohydride reduction glycosaminoglycans integrated pulsed amperometry     

Evaluation of Superoxide Scavenging Activities of Hamamelis Extract and Hamamelitannin

Hamamelitannin, which is a component of bark extract of hamamelis (Hamamelis virginior L.), was found to be a potent scavenger of superoxide anion radicals. Superoxide anion scavenging activity of the compound was evaluated by ESR-spin trap method using DMPO (5,5'-dimethyl-1-pyrroline-N-oxide) as a spin trapping agent. The IC₅₀ value (the concentration producing 50% inhibition of superoxide anion radicals) of hamamelitannin was found to be 1.38 ± 0.06 μM much lower than that of ascorbic acid (23.31 ± 2.23 μM). Supporting the superoxide scavenging activity of hamamelitannin, the compound showed both suppresive ability against depolymelization of hyaluronic acid and protective ability against cytotoxicity induced by superoxide anion radicals. Hamamelitannin increased the survival rate of fibroblast to 85.5 ± 3.3%, compared with that of control (27.2 ± 4.3%).     

Development of simulation models for protein folding in a thermal annealing process -I: a simulation of BPTI folding by the pearl necklace model

A model system is proposed to simulate the folding processesof proteins during thermal annealing. This system consists offour subsystems: (i) the pearl necklace model with isotropicinter-residue interactions; (ii) the extended pearl necklacemodel with anisotropic interaction potentials; (iii) moltenglobule phase dynamics; and (iv) final generation of the three-dimensionalstructure of a given protein. In this paper results obtainedwith the pearl necklace model are reported. This model consistsof spherical elements and virtual bonds of 3.8 Å in lengthand is intended to sinudate dynamical processes at relativelyhigh temperature where entropic terms play a dominant role.Inter-residue interactions are composed of spherical soft repulsivepotentials and hydrophobic interactions inherent to respectiveresidues. A simulation of folding processes of BPTI startingfrom the fully extended conformation indicated that intermediates,even at early stages of folding, are not randomly coiled butassume organized structures that resemble, to some extent, thenative conformation.     

Identification of photo-inactive phytochrome A in etiolated seedlings and photo-active phytochrome B in green leaves of the aurea mutant of tomato

The contents of spectrophotometrically measurable phytochrome A (PhyA) and phytochrome B (PhyB) and the corresponding immunochemically detectable apoproteins (PHYA and PHYB) were examined in dark- and light-grown tissues of the aurea mutant of tomato and its wild-type (WT). The amount of PHYA in etiolated aurea seedlings was found to be about 20% of that in the WT; this PHYA showed no photoreversible changes in absorbance, no downregulation of the level of PHYA in light-grown seedlings, and no differential proteolysis of Pr and Pfr species in vitro which was seen in the case of the WT. By contrast, the amount of PHYB in aurea seedlings was not significantly different from that in WT seedlings. Phytochrome isolated from green leaves of the aurea mutant and purified by ion-exchange chromatography showed a red/far-red reversible spectral change, and its elution profile during chromatography was essentially similar to that of PHYB. The results indicate that aurea is a mutant that is deficient in photoactive PhyA at the etiolated stage, when it contains a spectrally inactive PHYA. However, the mutant contains spectrally active PhyB in its green tissue as does the WT.