Genomic organization of the Neurospora crassa gsn gene: possible involvement of the STRE and HSE elements in the modulation of transcription during heat shock.

Glycogen synthase, an enzyme involved in glycogen biosynthesis, is regulated by phosphorylation and by the allosteric ligand glucose-6-phosphate (G6P). In addition, enzyme levels can be regulated by changes in gene expression. We recently cloned a cDNA for glycogen synthase ( gsn) from Neurospora crassa, and showed that gsn transcription decreased when cells were exposed to heat shock (shifted from 30 degrees C to 45 degrees C). In order to understand the mechanisms that control gsn expression, we isolated the gene, including its 5' and 3' flanking regions, from the genome of N. crassa. An ORF of approximately 2.4 kb was identified, which is interrupted by four small introns (II-V). Intron I (482 bp) is located in the 5'UTR region. Three putative Transcription Initiation Sites (TISs) were mapped, one of which lies downstream of a canonical TATA-box sequence (5'-TGTATAAA-3'). Analysis of the 5'-flanking region revealed the presence of putative transcription factor-binding sites, including Heat Shock Elements (HSEs) and STress Responsive Elements (STREs). The possible involvement of these motifs in the negative regulation of gsn transcription was investigated using Electrophoretic Mobility Shift Assays (EMSA) with nuclear extracts of N. crassa mycelium obtained before and after heat shock, and DNA fragments encompassing HSE and STRE elements from the 5'-flanking region. While elements within the promoter region are involved in transcription under heat shock, elements in the 5'UTR intron may participate in transcription during vegetative growth. The results thus suggest that N. crassa possesses trans -acting elements that interact with the 5'-flanking region to regulate gsn transcription during heat shock and vegetative growth.

Comparison of the genomes of two Xanthomonas pathogens with differing host specificities.

The genus Xanthomonas is a diverse and economically important group of bacterial phytopathogens, belonging to the gamma-subdivision of the Proteobacteria. Xanthomonas axonopodis pv. citri (Xac) causes citrus canker, which affects most commercial citrus cultivars, resulting in significant losses worldwide. Symptoms include canker lesions, leading to abscission of fruit and leaves and general tree decline. Xanthomonas campestris pv. campestris (Xcc) causes black rot, which affects crucifers such as Brassica and Arabidopsis. Symptoms include marginal leaf chlorosis and darkening of vascular tissue, accompanied by extensive wilting and necrosis. Xanthomonas campestris pv. campestris is grown commercially to produce the exopolysaccharide xanthan gum, which is used as a viscosifying and stabilizing agent in many industries. Here we report and compare the complete genome sequences of Xac and Xcc. Their distinct disease phenotypes and host ranges belie a high degree of similarity at the genomic level. More than 80% of genes are shared, and gene order is conserved along most of their respective chromosomes. We identified several groups of strain-specific genes, and on the basis of these groups we propose mechanisms that may explain the differing host specificities and pathogenic processes.

Molecular and biochemical characterization of the Neurospora crassa glycogen synthase encoded by the gsn cDNA.

Glycogen synthases catalyze the transfer of a glucosyl moiety from a nucleotide phosphosugar to a nascent glycogen chain via an alpha1-->4 linkage. Although many genes coding for glycogen synthases have been described, the enzymes from rabbit and yeast are the best characterized. The fungus Neurospora crassa accumulates glycogen during exponential growth, and mobilizes it at the onset of stationary phase, or when placed at high temperature or starved for carbon. Through a PCR methodology, the gsn cDNA coding for the N. crassa glycogen synthase was isolated, and the amino acid sequence of the protein was deduced. The product of the cDNA seems to be the only glycogen synthase present in N. crassa. Characterization of the gsn cDNA revealed that it codes for a 706-amino acids protein, which is very similar to mammalian and yeast glycogen synthases. Gene expression increased during exponential growth, reaching its maximal level at the end of the exponential growth phase, which is consistent with the pattern of glycogen synthase activity and glycogen level. Expression of the gsn is highly regulated at the transcriptional level. Under culture conditions that induce heat shock, conidiation, and carbon starvation, expression of the gsn gene was decreased, and glycogen synthase activity and glycogen content behaved similarly.

Biomass production and secretion of hydrolytic enzymes are influenced by the structural complexity of the nitrogen source in Fusarium oxysporum and Aspergillus nidulans.

The structural complexity of the nitrogen sources strongly affects biomass production and secretion of hydrolytic enzymes in filamentous fungi. Fusarium oxysporum and Aspergillus nidulans were grown in media containing glucose or starch, and supplemented with a nitrogen source varying from a single ammonium salt (ammonium sulfate) to free amino acids (casamino acids), peptides (peptone) and protein (gelatin). In glucose, when the initial pH was adjusted to 5.0, for both microorganisms, higher biomass production occurred upon supplementation with a nitrogen source in the peptide form (peptone and gelatin). With a close to neutrality pH, biomass accumulation was lower only in the presence of the ammonium salt. When grown in starch, biomass accumulation and secretion of hydrolytic enzymes (amylolytic and proteolytic) by Fusarium also depended on the nature of the nitrogen supplement and the pH. When the initial pH was adjusted to 5.0, higher growth and higher amylolytic activities were detected in the media supplemented with peptone, gelatin and casamino acids. However, at pH 7.0, higher biomass accumulation and higher amylolytic activities were observed upon supplementation with peptone or gelatin. Ammonium sulfate and casamino acids induced a lower production of biomass, and a different level of amylolytic enzyme secretion: high in ammonium sulfate and low in casamino acids. Secretion of proteolytic activity was always higher in the media supplemented with peptone and gelatin. Aspergillus, when grown in starch, was not as dependent as Fusarium on the nature of nitrogen source or the pH. The results described in this work indicate that the metabolism of fungi is regulated not only by pH, but also by the level of structural complexity of the nitrogen source in correlation to the carbon source.

High-level production of functional muscle alpha-tropomyosin in Pichia pastoris.

Although numerous studies have reported the production of skeletal muscle alpha-tropomyosin in E. coli, the protein needs to be modified at the amino terminus in order to be active. Without these modifications the protein does not bind to actin, does not exhibit head-to-tail polymerization, and does not inhibit the actomyosin Mg(2+)-ATPase in the absence of troponin. On the other hand, the protein produced in insect cells using baculovirus as an expression vector (Urbancikova, M., and Hitchcock-DeGregori, S. E., J. Biol. Chem., 269, 24310-24315, 1994) is only partially acetylated at its amino terminal and therefore is not totally functional. In an attempt to produce an unmodified functional recombinant muscle alpha-tropomyosin for structure-function correlation studies we have expressed the chicken skeletal alpha-tropomyosin cDNA in the yeast Pichia pastoris. Recombinant protein was produced at a high level (20 mg/L) and was similar to the wild type muscle protein in its ability to polymerize, to bind to actin and to regulate the actomyosin S1 Mg(2+)-ATPase.

Efficacy of interferon-based therapy in the treatment of thalassaemic patients with chronic hepatitis C: a meta-analysis.

OBJECTIVE: To identify the interferon-alpha (IFNalpha) treatment protocol most suitable for patients with thalassaemia major who have chronic hepatitis C. DESIGN AND SETTING: This was a meta-analysis of studies in the international literature between 1990 and 1999. METHODS: Studies were identified from a search of Medline and Embase, and analysed by the Mantel-Haenszel-Peto statistical method. RESULTS: We identified 6 nonrandomised trials, 2 of which were controlled, that treated a total of 201 patients. Most studies used the lowest dose level (3 MIU/m(2)), all used a thrice-weekly regimen, and most used IFNalpha-2b, although the use of natural IFNalpha did not induce production of anti-interferon antibodies. The best sustained response and remission rates tended to be achieved with higher doses and longer cycles of IFNalpha. CONCLUSIONS: The best interferon-based therapy to treat polytransfused thalassaemic patients with chronic hepatitis C is represented by the use of natural IFNalpha or IFNalpha-2b, initially at high dosages (5 to 10 MIU/m(2) 3 times weekly) for 6 months, followed by lower dosages (3 MIU/m(2) 3 times weekly) for a further 6 to 9 months.

Expression and characterization of Geotrichum candidum lipase I gene. Comparison of specificity profile with lipase II.

Despite tremendous progress in the elucidation of three-dimensional structures of lipases, the molecular basis for their observed substrate preference is not well understood. In an effort to correlate the lipase structure with its substrate preference and to clarify the contradicting reports in the literature, we have compared the enzymic characteristics of two closely related recombinant lipases from the fungus Geotrichum candidum. These enzymes were expressed in the yeast Saccharomyces cerevisiae as fusions with an N-terminal poly(His) tag and were purified in a single step by metal-affinity chromatography. Their specific activities against a series of triacylglycerol substrates were compared using a titrimetric assay. The substrates varied in fatty acyl chain length, number of double bonds and their position along the chain. G. candidum lipases I and II (GCL I and GLC II) are markedly different with respect to their substrate preferences. For unsaturated substrates having long fatty acyl chains (C18:2 cis-9, cis-12 and C18:3 cis-9, cis-12, cis-15), GCL I showed higher specific activity than GCL II, whereas GCL II showed higher specific activity against saturated substrates having short fatty acid chains (C8, C10, C12 and C14). We have constructed a hybrid molecule containing the N-terminal portion of GCL I (including the flap covering the active site) linked to the C-terminal portion of GCL II. The hybrid molecule showed a substrate preference pattern identical to that of GCL II. These results indicate that sequence variation within the N-terminal 194 amino acids of G. candidum lipases do not contribute to the observed variation in efficiency by which the lipases hydrolyze their substrates. Moreover, it also shows that the flap region in GCL is not directly involved in substrate differentiation, even though this region is thought to be involved in recognition of the interface and in the activation of the enzyme.

Polymorphism in the lipase genes of Geotrichum candidum strains.

The fungus Geotrichum candidum produces extracellular lipases. Purification and characterization of different lipase isoforms from various G. candidum strains is difficult due to the close physical and biochemical properties of the isoforms. Consequently, the characterization of these enzymes and their substrate specificities has been difficult. We have determined the lipase genes present in four strains of G. candidum (ATCC 34614, NRCC 205002, NRRL Y-552 and NRRL Y-553) by molecular cloning and DNA sequencing. Each strain contains two genes similar to the previously identified lipase I and lipase II cDNAs. Our data suggest that no other related lipase genes are present in these strains. Each lipase-gene family shows sequence variation (polymorphism) that is confirmed by Southern-blot analysis. This polymorphism and the sequence differences between lipase I and lipase II have been localized within the previously determined three-dimensional structure of lipase II. Although most of the amino acid substitutions are located on the protein surface, some are present in structural features possibly involved in determining substrate specificity.

New amylolytic yeast strains for starch and dextrin fermentation.

Yeast strains capable of fermenting starch and dextrin to ethanol were isolated from samples collected from Brazilian factories in which cassava flour is produced. Considerable alcohol production was observed for all the strains selected. One strain (DI-10) fermented starch rapidly and secreted 5 times as much amylolytic enzyme than that observed for Schwanniomyces alluvius UCD 54-83. This strain and three other similar isolates were classified as Saccharomyces cerevisiae var. diastaticus by morphological and physiological characteristics and molecular taxonomy.

In vivo transfer of delayed hypersensitivity to Trypanosoma cruzi antigens with polysomal immune RNA.

The polysomal immune RNA (iRNA) was extracted from the spleens of mice infected with Trypanosoma cruzi. The RNA isolated from normal (nRNA) animals served as control. We found that the polysomal iRNA is able to transfer delayed-type hypersensitivity (DTH) in vivo to T. cruzi antigens as assessed by the inhibition of cell migration assay and skin test. We also demonstrated that this phenomenon is antigen specific. The polysomal iRNA preparations were fractionated by affinity chromatography on oligo (dT)-cellulose column. Our results suggest that the poly (A)-containing iRNA is the fraction responsible for transferring DTH in vivo to T. cruzi antigens.

Poly(A)-containing RNA from the spleens of mice with Chagas' disease triggers in vitro macrophage resistance to Trypanosoma cruzi.

Mouse peritoneal macrophages exposed to the RNA from the spleens of mice infected with Trypanosoma cruzi (iRNA) exhibit enhanced resistance to this parasite. The poly(A)-containing iRNA was found to be the active fraction. No such activity was observed in macrophages incubated with RNA from normal mice (nRNA) or with synthetic poly A.

The protective effect of the 4-5S immune RNA against Trypanosoma cruzi infection in mice.

The immune RNA (iRNA) was extracted from the spleens of mice infected with Trypanosoma cruzi. The 4-5S iRNA, obtained by sucrose gradient centrifugation, was effective in reducing the parasitemia and mortality of mice challenged with a virulent strain of T. cruzi. No such activity was found in the RNA fraction obtained from the spleens of normal (4-5S nRNA) animals. Mouse peritoneal macrophages exposed to the 4-5S iRNA were able to inhibit the growth of T. cruzi. These results suggest that the protective effect of the 4-5S iRNA against T. cruzi infection could be due to its ability in enhancing macrophage resistance to this parasite.

In vitro transfer of reactivity to Trypanosoma cruzi antigens from rat cells to human cells with immune RNA.

The immunologic activity of polysomal RNA isolated from the spleens of rats infected with Trypanosoma cruzi was assessed by the leukocyte migration-inhibition assay as an in vitro correlate of delayed hypersensitivity. This RNA preparation transferred reactivity to T. cruzi antigens to nonsensitized human lymphocytes from peripheral blood. This transfer of reactivity was abolished by treatment of the RNA preparation with ribonuclease. The transfer of delayed hypersensitivity in vitro from rat cells to human cells was antigen-specific. Polysomal RNA also conferred significant protection against infection with T. cruzi in mice, as evaluated by the level of parasitemia and the survival rate of mice challenged with virulent strain of Y of T. cruzi.

Fractionation of immune RNA isolated from the spleens of mice infected with Trypanosoma cruzi.

Immune RNA was extracted from the spleens of mice infected with Trypanosoma cruzi. The immunologic activity of crude immune RNA and the fractions that were obtained by sucrose density gradient centrifugation was assessed by the in vitro macrophage migration-inhibition assay. Crude immune RNA can convert nonsensitized mouse peritoneal cells to a state of specific immunologic reactivity to T. cruzi antigens. The transfer activity of crude immune RNA was abolished by treatment with pancreatic ribonuclease. The 4S-5S RNA was the only fraction active in transferring delayed hypersensitivity in experimental Chagas' disease; however, the 4S transfer RNA also isolated from the spleens of mice with Chagas' disease has no immunologic activity. Immune RNA in experimental Chagas' disease may be an informational RNA.