The role of CRE1 in nucleosome positioning within the cbh1 promoter and coding regions of Trichoderma reesei.

Nucleosome positioning within the promoter and coding regions of the cellobiohydrolase-encoding cbh1 gene of Trichoderma reesei was investigated. T. reesei is a filamentous fungus that is able to degrade dead plant biomass by secreting enzymes such as cellulases, a feature which is exploited in industrial applications. In the presence of different carbon sources, regulation of one of these cellulase-encoding genes, cbh1, is mediated by various transcription factors including CRE1. Deletion or mutation of cre1 caused an increase in cbh1 transcript levels under repressing conditions. CRE1 was shown to bind to several consensus recognition sequences in the cbh1 promoter region in vitro. Under repressing conditions (glucose), the cbh1 promoter and coding regions are occupied by several positioned nucleosomes. Transcription of cbh1 in the presence of the inducer sophorose resulted in a loss of nucleosomes from the coding region and in the re-positioning of the promoter nucleosomes which prevents CRE1 from binding to its recognition sites within the promoter region. Strains expressing a non-functional CRE1 (in strains with mutated CRE1 or cre1-deletion) exhibited a loss of positioned nucleosomes within the cbh1 coding region under repressing conditions only. This indicates that CRE1 is important for correct nucleosome positioning within the cbh1 coding region under repressing conditions.

Profiling CpG island field methylation in both morphologically normal and neoplastic human colonic mucosa.

Aberrant CpG island (CGI) methylation occurs early in colorectal neoplasia. Quantitative methylation-specific PCR profiling applied to biopsies was used to quantify low levels of CGI methylation of 18 genes in the morphologically normal colonic mucosa of neoplasia-free subjects, adenomatous polyp patients, cancer patients and their tumours. Multivariate statistical analyses distinguished tumour from mucosa with a sensitivity of 78.9% and a specificity of 100% (P=3 x 10(-7)). In morphologically normal mucosa, age-dependent CGI methylation was observed for APC, AXIN2, DKK1, HPP1, N33, p16, SFRP1, SFRP2 and SFRP4 genes, and significant differences in CGI methylation levels were detected between groups. Multinomial logistic regression models based on the CGI methylation profiles from normal mucosa correctly identified 78.9% of cancer patients and 87.9% of non-cancer (neoplasia-free+polyp) patients (P=4.93 x 10(-7)) using APC, HPP1, p16, SFRP4, WIF1 and ESR1 methylation as the most informative variables. Similarly, CGI methylation of SFRP4, SFRP5 and WIF1 correctly identified 61.5% of polyp patients and 78.9% of neoplasia-free subjects (P=0.0167). The apparently normal mucosal field of patients presenting with neoplasia has evidently undergone significant epigenetic modification. Methylation of the genes selected by the models may play a role in the earliest stages of the development of colorectal neoplasia.

The procyanidin-mediated induction of apoptosis and cell-cycle arrest in esophageal adenocarcinoma cells is not dependent on p21(Cip1/WAF1).

Previous studies have shown that the proanthocyanidin-mediated induction of apoptosis and arrest of the cell cycle in cancer cells was associated with up-regulation of p21(Cip1/WAF1) (p21), suggesting that p21 may be the molecular mediator of the observed effects. Here we show that procyanidins induce a rapid and sustained arrest of the cell cycle, and increase apoptosis, concomitant with an increase in p21 expression. However, blocking the PA-induced up-regulation of p21 expression with siRNA did not alter PA-mediated changes in apoptosis and cell cycle, demonstrating that p21 is not responsible for the PA-induced effects.

Methylation of the ESR1 CpG island in the colorectal mucosa is an 'all or nothing' process in healthy human colon, and is accelerated by dietary folate supplementation in the mouse.

ESR1 is frequently silenced by CGI (CpG island) methylation, both in human colorectal tumours and, in an age-dependent manner, in healthy mucosa. It is not clear, however, whether methylation of individual cytosines occurs randomly within the epithelial genome, or preferentially within individual cells as an 'all-or-nothing' phenomenon. CGI methylation can be quantified in human DNA residues recovered from faecal samples. We used bisulphite genomic sequencing of human DNA from this source and from a colorectal cancer cell line (SW48) to show that the ESR1 CGI is methylated in an allele-specific manner. This provides support for the 'all or none' mechanism for methylation of this gene, and shows how age-dependent methylation of the ESR1 CGI leads rapidly to silencing of the gene within the cells, and hence the colonic crypt within which it occurs. Preliminary studies with a rodent model suggest the rate of age-dependent methylation of ESR1 is modifiable by dietary folate.

Use of a histone H4 promoter to drive the expression of homologous and heterologous proteins by Penicillium funiculosum.

Two genes encoding histone H4 (H4.1 and H4.2) from Penicillium funiculosum have been cloned and characterised. Structurally, the histone H4.1 gene is divergently linked to the histone H3 gene and the two genes are separated by approximately 800 bp. The transcription of the histone H4.1 and H4.2 genes in P. funiculosum appears to be distinctively regulated. Histone H4.1 mRNA showed a high steady-state level during the early stages of batch culture that decreased as growth reached the stationary phase. In contrast, the expression of the histone H4.2 gene was lower than that of H4.1 throughout batch growth and increased gradually with time. In order to expand the industrial application of P. funiculosum as a host for the production of heterologous proteins, the promoter of the histone H4.1 gene was successfully used to drive the expression of an intracellular bacterial enzyme, beta-glucuronidase, and a secreted homologous enzyme, xylanase C. The constitutive secretion of xylanase C was achieved in the absence of other xylanases by batch fermentation in the presence of glucose.

A modular esterase from Penicillium funiculosum which releases ferulic acid from plant cell walls and binds crystalline cellulose contains a carbohydrate binding module.

An esterase was isolated from cultures of the filamentous fungus Penicillium funiculosum grown on sugar beet pulp as the sole carbon source. The enzyme (ferulic acid esterase B, FAEB) was shown to be a cinnamoyl esterase (CE), efficiently releasing hydroxycinnamic acids from synthetic ester substrates and plant cell walls, and bound strongly to microcrystalline cellulose. A gene fragment was obtained by PCR using partial amino-acid sequences obtained from the pure enzyme and used to a probe a P. funiculosum genomic DNA library. A clone containing a 1120-bp ORF, faeB, was obtained which encoded a putative 353-residue preprotein including an 18-residue signal peptide, which when expressed in Eschericia coli produced CE activity. Northern analysis showed that transcription of faeB was tightly regulated, being stimulated by growth of the fungus on sugar beet pulp but inhibited by free glucose. The faeB promoter sequence contains putative motifs for binding an activator protein, XLNR, and a carbon catabolite repressor protein, CREA. FAEB was comprised of two distinct domains separated by a 20 residue Thr/Ser/Pro linker region. The N-terminal domain comprised 276 amino acids, contained a G-X-S-X-G motif typical of serine esterases, and was shown to be a member of a family comprising serine esterases, including microbial acetyl xylan esterases, poly (3-hydroxyalkanoate) depolymerases and CEs, and proteins of unknown function from Mycobacterium spp. and plants. The C-terminal domain comprised 39 amino acids and closely resembled the family 1 cellulose binding carbohydrate-binding modules (CBM) of fungal glycosyl hydrolases. This is the first report of a fungal CE with a CBM.

Trichoderma reesei sequences that bind to the nuclear matrix enhance transformation frequency.

Three DNA fragments, trs1, 2 and 3, were isolated from the Trichoderma reesei genome on the basis of their ability to promote autonomous replication of plasmids in Saccharomyces cerevisiae. Each trs element bound specifically to the isolated T. reesei nuclear matrix in vitro, and two of them bound in vivo, indicating that they are matrix attachment regions (MARs). A similar sequence previously isolated from Aspergillus nidulans (ans1) was also shown to bind specifically to the T. reesei nuclear matrix in vitro. The T. reesei MARs are AT-rich sequences containing 70%, 86% and 73% A + T over 2.9, 0.8 and 3.7 kb, respectively for trs1, 2 and 3. They exhibited no significant sequence homology, but were shown to contain a number of sequence motifs that occur frequently in many MARs identified in other eukaryotes. However, these motifs occurred as frequently in the trs elements as in randomly generated sequences with the same A + T content. trs1 and 3 were shown to be present as single copies in the T. reesei genome. The presence of the trs elements in transforming plasmids enhanced the frequency of integrative transformation of T. reesei up to five fold over plasmids without a trs. No evidence was obtained to suggest that the trs elements promoted efficient replication of plasmids in T. reseei. A mechanism for the enhancement of transformation frequency by the trs elements is proposed.

Specificity of the binding domain of glucoamylase 1.

Glucoamylase 1 from Aspergillus niger hydrolyses granular starch at an increased rate due to the presence of a C-terminal starch-binding domain. This domain was isolated and shown to bind to the malto-oligosaccharides Glc2 to Glc11 with a stoichiometry of 1 mol ligand/mol protein. The affinity for these ligands increased with increasing degree of polymerisation until Glc9, above which no further increase was observed. We suggest that this indicates that for maximum affinity the substrate should be able to form a helical conformation, which mimics the conformation of amylose in granular starch. We propose a model of how the complex between the malto-oligosaccharides and the binding domain is formed and indicate how this affects the differences in binding modes for soluble and insoluble substrates. Glucono-1,5-lactone interacts with the binding domain at a different site to the malto-oligosaccharides allowing the formation of a ternary complex between the binding domain, a malto-oligosaccharide and glucono-1,5-lactone. The binding domain also binds to linear alpha-1,6-linked glucose digosaccharides (dextran), but with much lower affinity than for alpha-1,4-linked glucose. This ligand appears to interact with the binding domain at both binding sites, i.e. at the site to which the malto-oligosaccharides bind and also at the site to which glucono-1,5-lactone binds. The relevance of the results to the mechanism of action of other polysaccharide-hydrolysing enzymes containing both a catalytic and a binding domain is discussed.

O-glycosylation and stability. Unfolding of glucoamylase induced by heat and guanidine hydrochloride.

We have examined the stabilities of the catalytic and binding domains of glucoamylase 1 from Aspergillus niger and how these stabilities are affected by the O-glycosylated linker glycopeptide which separates the domains. On heating, the catalytic domain unfolds irreversibly, whereas the binding domain unfolds reversibly as shown by differential scanning calorimetry and by 1H NMR. The stability of three functional peptides, derived from glucoamylase 1, containing the binding domain alone and with 10 or 38 residues of the linker glycopeptide [Williamson, G., Belshaw, N.J. and Williamson, M. (1992) Biochem. J. 282, 423-428] was examined. Refolding in each case was reversible after thermal or chemical denaturation. beta-Cyclodextrin stabilised the binding domain by the same amount when it was part of glucoamylase 1 or an isolated domain. The thermal stability of the catalytic domain was not affected by the binding domain; however, the catalytic domain increased the melting temperature of the binding domain. Furthermore, the linker glycopeptide stabilised the binding domain against reversible thermal and chemical denaturation by about 10 kJ/mol, but only a portion of the O-glycosylated residues were required for stabilisation. On a simple molecular mass basis, the linker glycopeptide does not contribute as much as expected to the denaturational enthalpy of glucoamylase 1 and, in addition, shows only a small conformational change on chemical or thermal denaturation; this supports an extended structure for the linker. The results demonstrate that the unfolding pathway of glucoamylase 1 depends on the concentration of beta-cyclodextrin and that the presence of the catalytic domain and/or the linker glycopeptide stabilises the binding domain.

O-glycosylation in Aspergillus glucoamylase. Conformation and role in binding.

Functional peptides have been produced by proteolysis of glucoamylase (glucan 1,4-alpha-glucosidase; EC 3.2.1.3) from Aspergillus niger and purified by affinity chromatography, gel filtration and two ion-exchange-chromatography steps. The peptides correspond to residues 499-616 and 509-616 of the original glucoamylase molecule. Together with G1C (residues 471-616 from glucoamylase 1) [Belshaw & Williamson (1990) FEBS Lett. 269, 350-353], the three peptides all contain the C-terminal domain (residues 509-616) but, in addition, contain different proportions of the O-glycosylated region. The properties of these peptides have been compared to define the function of the O-linked oligosaccharides in this protein. The O-glycosylated region plays only a minor role in binding to hydrogen-bond ordered starch. The difference between the apparent free energy (delta G) for binding between the non-glycosylated C-terminal domain (-26.0 kJ/mol) and the C-terminal domain containing the fully O-glycosylated region (-25.0 kJ/mol) is only 1.0 kJ/mol. Binding to beta-cyclodextrin suggests that even this difference may reflect a small conformational change in the C-terminal domain rather than a direct effect of the O-linked sugars. The c.d. spectrum of the O-glycosylated region is deduced by comparison of the three peptides and is predominantly that of a random-coil structure. Two-dimensional n.m.r. spectra of glucoamylase and of the glycosylated peptide 499-616 show that the binding domain is more mobile than the catalytic domain and that its mobility is further increased on removal of the catalytic domain. The O-glycosylated region is more mobile still, and there is a marked increase in its mobility on removal of the catalytic domain. The O-glycosylated region in the intact protein can therefore be envisaged as a semi-rigid rod. The results show that a major function of O-glycosylation in glucoamylase 1 is to provide an extended peptide backbone and hence a fixed distance in linking the catalytic and binding domains. It does not in itself significantly increase the binding affinity for starch.

Interaction of beta-cyclodextrin with the granular starch binding domain of glucoamylase.

The granular starch binding domain of glucoamylase 1 (EC 3.2.1.3 1,4-alpha-D-glucan glucohydrolase) binds two molecules of beta-cyclodextrin, with a dissociation constant (Kd) for the second ligand of 1.68 microM. The catalytic domain showed no interaction with beta-cyclodextrin. Beta-cyclodextrin competitively inhibited the adsorption of the binding domain onto granular starch with an inhibition constant (Ki) of 11.0 +/- 1.9 microM. The results show that beta-cyclodextrin binds to the binding domain of glucoamylase at the same site(s) as granular starch.

Production and purification of a granular-starch-binding domain of glucoamylase 1 from Aspergillus niger.

A domain of glucoamylase 1 from Aspergillus niger which binds to granular starch was produced by proteolytic digestion and purified to apparent homogeneity by extraction with corn starch followed by anion-exchange chromatography and gel filtration. The peptide has a molecular weight of 25,100, contains approximately 38% carbohydrate (w/w) and corresponds to residues 471-616 at the C-terminus of glucoamylase 1. The peptide bound to granular corn starch maximally at 1.08 nmol/mg starch. It inhibited the hydrolysis of granular starch by glucoamylase 1 but had no effect on the hydrolysis of starch in solution.

The effect of cadaverine on the formation of anabasine from lysine in hairy root cultures of Nicotiana hesperis.

Unlabelled cadaverine did not diminish the incorporation into anabasine of (14)C from L-[U-(14)C] lysine supplied to hairy root cultures of Nicotiana nesperis, despite causing a stimulation of anabasine production. The finding is discussed in the context of previous observations indicating that free cadaverine is not an intermediate in the biosynthesis of anabasine from lysine.