Improvement of gene targeting in Aspergillus nidulans with excess non-homologous fragments.

Efficiency of gene targeting was increased more than 10-fold when an excess molar ratio of non-homologous fragments was added in the transformation of Aspergillus nidulans. In the targeted transformants, integration of such fragments into the host chromosome was a rare event. Hence, our approach proved practical in terms of producing successful targeting events without disturbing the host chromosomes.

The occurrence of eukaryotic type III glutamine synthetase in the marine diatom Chaetoceros compressum.

Glutamine synthetase (GS) has been described as one of the oldest functioning genes and thus a good molecular clock protein. GS is diverged into three distinct forms, type I (GSI), type II (GSII) and type III (GSIII), the last type of which is a member of the most recently discovered family among GSs and thus has been reported from a limited number of prokaryotes. In the present study, we determined the full-length sequence of GSIII from the marine diatom Chaetoceroscompressum. The 3' untranslated region of the diatom GSIII gene was composed of a polyadenylation signal followed by a poly (A)(+) tail, clearly demonstrating that its mRNA is transcribed from the eukaryotic genome. We also screened available genome databases and identified full-length GSIII sequences from 5 eukaryotic species. These eukaryotic GSIIIs specifically contained regions A-D and a long additional sequence flanking region V toward the C-terminal site, both being specific to GSIII. Phylogenic analysis revealed that eukaryotic GSIIIs are not within a monophyletic relationship with the possible occurrence of lateral gene transfer in GSIII during evolution.

Comparison between polyethylene glycol- and polyethylenimine-mediated transformation of Aspergillus nidulans.

Genetic transformation of many filamentous fungi is carried out by a protocol that utilizes polyethylene glycol (PEG) and calcium ion (Ca2+). This method has remained practically unchanged for more than 20 years, but the roles these molecules play are not definitively understood. To gain a better understanding, we have compared PEG transformation to a protocol using polyethylenimine (PEI) that is the basis for non-viral transfection in mammals and which has a well established molecular model for assisting DNA uptake. Protoplasts of Aspergillus nidulans could be transformed in the presence of Ca2+ with a relatively high ratio of PEI to DNA molecules. By comparing PEI and PEG in terms of interaction with DNA, fungal protoplasts, and response to different transformation conditions, we propose that the role of PEG is most likely to function after transforming DNA is incorporated into protoplasts, rather than the accepted view that it functions outside of the cell. Confirmation that protoplast fusion was not involved in DNA uptake is consistent with this hypothesis.

Molecular characterization of Japanese sillago vitellogenin and changes in its expression levels on exposure to 17beta-estradiol and 4-tert-octylphenol.

We cloned a full-length cDNA encoding vitellogenin (VTG) from a marine teleost, the Japanese sillago Sillago japonica. The cloned sillago VTG contained signal peptide, lipovitellin heavy chain, phosvitin, lipovitellin light chain, and beta'-component in the order from the N-terminus. An exposure to 17beta-estradiol significantly increased the levels of plasma VTG, but not hepatic VTG mRNA in males. Neither plasma VTG nor hepatic VTG mRNA levels were affected by the exposure to 4-tert-octylphenol. Hepatic VTG mRNA levels in males increased at 1 day after intraperitoneal administration of 17beta-estradiol but decreased in the subsequent 5 days. However, plasma VTG levels remained high for 5 days after administration, suggesting that the accumulation period of plasma VTG is longer than that of hepatic VTG mRNA in males. Therefore, VTG mRNA may be a suitable indicator of temporal exposure to estrogenic chemicals in the environment, whereas plasma VTG is useful to detect consecutive exposure.

The inhibitory effect of ouabain on the response of slowly adapting pulmonary stretch receptors to hyperinflation in the rabbit.

The combined effects of ouabain (Na(+)-K(+) ATPase inhibitor) and hyperinflation (inflation volume=three tidal volumes) on slowly adapting pulmonary stretch receptors (SARs) were studied before and after administration of nifedipine (an L-type Ca(2+) channel blocker) and KB-R7943 (a reverse-mode Na(+)-Ca(2+) exchanger blocker) in anesthetized, artificially ventilated rabbits after bilateral vagotomy. Before ouabain administration, hyperinflation stimulated SAR activity. After 20 min of ouabain administration (30 microg/kg) the SARs increased discharge rates in normal inflation. Under these conditions, hyperinflation initially stimulated SAR activity but subsequently inhibited the activity at peak inflation. Additional administration of 60 microg/kg ouabain (total dose=90 microg/kg) caused a further stimulation of SAR activity, but 20 min later both normal inflation and hyperinflation resulted in a greater inhibition of the receptor activity. The hyperinflation-induced SAR inhibition in the presence of ouabain (30 microg/kg) was not significantly altered by administration of either nifedipine (2 and 4 mg/kg) or KB-R7943 (1 and 3 mg/kg). In another series of experiments, we further examined the combined effects of ouabain and hyperinflation in veratridine (a Na(+) channel opener, 40 microg/kg)-treated animals. After recovery from the veratridine effect on SAR activity, which vigorously stimulated the receptor activity, ouabain treatment (30 microg/kg) that silenced the receptor activity at peak inflation greatly inhibited hyperinflation-induced SAR stimulation. These results suggest that hyperinflation-induced SAR inhibition in the presence of ouabain may be related to a Na(+) overload, but not to a Ca(2+) influx via activation of L-type Ca(2+) channels, in the SAR endings.

An appropriate increase in the transcription of Aspergillus nidulans uvsC improved gene targeting efficiency.

Gene targeting to knock out the activity of specific genes has become important due to recent progress in genomics research. But this technique is still unavailable for many organisms, including economically important microorganisms, due to the high background of ectopic integration during genetic transformation. Strategies to improve targeting efficiency have included manipulating the expression of genes that are involved in homologous recombination. In this study, transcription of Aspergillus nidulans uvsC was elevated using the promoter sequences of the glyceraldehyde-3-phosphate dehydrogenase and Taka-amylase A genes from A. nidulans and A. oryzea respectively. Although a several-fold increase in the efficiency of targeting was observed at 3 loci, mycelial growth was suppressed in strains that had higher levels of uvsC transcription. These results suggest that uvsC is a rate-limiting factor in gene targeting, and that the increased efficiency of this targeting is hindered by a negative effect of increased transcription on cell proliferation.

Suppression of tandem-multimer formation during genetic transformation of the mycotoxin-producing fungus Penicillium paxilli by disrupting an orthologue of Aspergillus nidulans uvsC.

An orthologue of Aspergillus nidulans uvsC and Saccharomyces cerevisiae RAD51 was cloned from the filamentous fungus, Penicillium paxilli. A mutation in uvsC causes UV sensitivity during germination. The product of RAD51 is involved in meiotic recombination and DNA damage repair. The deduced amino acid sequence of the product of this gene (Pprad51) shared 92% identity with UVSC. Site-specific disruption of pprad51 showed a significant effect for extra-cellular DNA integration. Transformation of the null mutant with pII99, which confers geneticin resistance, resulted in a shift from a predominance of direct repeats at a single site to single copies when compared with a control strain. A copy-number effect of integrated pII99 for geneticin selection was suggested as the frequency of direct repeat formation was less when selected at a lower concentration in the control strain. However, such an effect was not observed in the null mutant, further supporting an involvement of Pprad51 in direct repeat formation.

[A case of tuberculosis pleuritis developing contralateral pleural effusion during anti-tuberculosis chemotherapy, falling into chronic respiratory failure].

The patient was a 74 year-old male presenting right pleural effusion with mild fever. His temperature was 37.0 degrees C. Culture of a pleural biopsy specimen revealed Mycobacterium tuberculosis, although culture of sputum and pleural effusion were negative. Therapy was begun with 300 mg of isoniazid (INH) per day, 600 mg of rifampicin (RFP) per day, and 1200 mg of pyrazinamide (PZA) per day. His temperature improved temporarily. One week after beginning of the therapy he had a fever over 38.0 degrees C. On the 17th day after starting chemotherapy, a chest radiological examination showed left pleural effusion in which numerous lymphocytes were found but Mycobacterium tuberculosis was negative. We assumed that the left pleural effusion was due to a paradoxical reaction to the anti-tuberculosis chemotherapy. After 3 days' discontinuation, the same regimen was resumed with an addition of prednisolone, but bilateral pleural effusion remained and the case finally fell into chronic respiratory failure.