Efficient integrative transformation of the phytopathogenic fungus Alternaria alternata mediated by the repetitive rDNA sequences

An attempt was made to transform Alternaria alternata protoplasts using a plasmid vector, pDH25, bearing the Escherichia coli hygromycin B (Hy) phosphotransferase gene (hph) under the control of the Aspergillus nidulans trpC promoter. Transformants arose on a selective medium containing 100 μg Hy/ml. There were two types of transformants, forming large and small colonies on the selective medium. Transformation with one μg of the vector produced an average of 4.5 large colonies and 600 small ones. In large-colony transformants, the vector often integrated into the recipient chromosome in the form of highly rearranged tandem arrays. To increase transformation efficiency, fragments of the highly repetitive ribosomal RNA gene cluster (rDNA) of A. alternata were used to construct four new vectors for homologous recombination system. Use of these vectors gave higher transformation efficiency than the original plasmid. The best vector, pDH25r1a, gave rise to large-colony transformants at a frequency 20 times higher than pDH25. Transformation events in A. alternata with pDH25r1a occured by homologous recombination as a single crossover between the plasmid-borne rDNA segment and its homologue in the chromosome, often giving rise to tandemly repeated vector DNA.     

Regulated Expression of a Gene-Fusion Product Derived from the Gene for Phytochrome I from Pisum sativum and the uidA Gene from E. coli in Transgenic Petunia hybrida

The 5'-upstream region (2.4 kb) of the gene for phytochromeI from Pisum sativum (phyl) was fused to the uidA gene fromEscherichia coli that encodes ß-glucuronidase (GUS).The resulting PHY-GUS fusion was introduced into Petunia hybridaand was used as a reporter of the expression of the phyI genewhich was recognized by GUS activity. The PHY-GUS fusion wasexpressed at a relatively high level when transgenic plantswere grown in the dark, while leaves and stems of light-grownplants showed background activity. Flowers of light-grown plantswere shown to have significant levels of GUS activity but rootsdid not have such activity. When light-grown transgenic plantswere transferred to the dark, they expressed the activity atlevels that corresponded to those of dark-grown plants. Lighttreatment prior to growth in darkness revealed red/far-red reversibilityof recovery of the activity. Thus, the 2.4-kb fragment fromthe 5' region of the phyI gene carries the information necessaryfor the light-repressible autoregulation. (Received March 30, 1991; Accepted May 20, 1991)     

Serum-dependent regulation of proliferation of cultured rat fibroblasts in G1 and G2 phases

We reported that: (i) 3Y1tsF121 cells, a temperature-sensitive (ts) mutant of rat 3Y1 fibroblasts, are reversibly arrested either in the G1 or in the G2 phase, at the nonpermissive temperature. (ii) Cells retain the ability to resume proliferation at the permissive temperature after prolonged arrest in the G1 phase (for 5 days), whereas they lose it after prolonged arrest in the G2 phase (over 24 h). (iii) The G1 arrest is overcome at the nonpermissive temperature by the addition of fresh serum (H. Zaitsu and G. Kimura (1984) J. Cell. Physiol. 119, 82; (1985) J. Cell. Physiol. 124, 177). In the present study, the G2 arrest was overcome by exposing the cells to fresh serum, at the nonpermissive temperature. The G2 arrest occurred only at a higher cell density than that of the G1 arrest. The efficiency of the overcome was higher in the case of the G2 arrest than in case of the G1 arrest. When cells synchronized at the G1/S border by aphidicolin at the permissive temperature were released from the block, they divided in the absence of serum, at the permissive temperature. Even if they had passed through the previous G2 phase in a very high concentration of fresh serum at the permissive temperature, mitotic cells did not enter the S phase in the absence of serum, even at the permissive temperature. When the cells arrested in the G1 phase (not in G0) due to the ts defect were incubated in the absence of serum at the permissive temperature, only 34% entered the S phase and only 15% divided. These results suggest that (i) the ts defect in 3Y1tsF121 limiting cellular proliferation in both the G1 and the G2 phases is probably due to a single mutational event, and is a serum-requiring event. (ii) Preparation of the serum-requiring event which is required for the G2 traverse is completed in the G1 phase, under ordinary conditions. (iii) However, cells are able to fulfill the serum-requiring event in the G2 phase as well as in the G1 phase when the preparation is below the required level. (iv) The commitment to DNA synthesis is not necessarily a commitment to cell division. (v) Cells are arrested in the G1 phase more safely and more effectively than in the G2 phase, by the serum-related mechanism.     

A new species and a new subspecies of the stargazer genusGnathagnus from Northwestern Australia

Heretofore the genusGnathagnus (Uranoscopidae) has been composed of three species:G. elongatus (Temminck et Schlegel) from northern Australia to Japan,G. innotabilis (Waite) from Australasia, andG. egregius (Jordan et Thompson) from the western Atlantic Ocean. In this paper, a new species and a new subspecies from northwestern Australia are described and a key to the known species and subspecies of the genus is presented.     

Termination of endothelin signaling: Role of nitric oxide

Cellular mechanisms responsible for the termination of ET-1 signal are poorly understood. In order to examine the hypothesis that nitric oxide serves as a physiological brake of ET- 1 signaling, Chinese hamster ovary (CHO) cells stably transfected with the ET_A receptor cDNA (CHO-ET) were studied. CHO-ET responded to ET-1 with robust [Ca²⁺], transients and developed a long-lasting homologous desensitization. Donors of nitric oxide (NO), 3-morpholino-sydnonimine HCl(SIN-1), or sodium nitroprusside (SNP) reduced the amplitude of these responses, accelerated the rate of [Ca²⁺], recovery, and counteracted the development of homologous desensitization by a cyclic GMP-independent mechanism, suggesting an alternative mode for NO modulation of ET-1 responses. Stimulation of CHO-ET cells with mastoparan, a wasp venom acting directly on G proteins (bypassing receptor activation), was inhibited by NO, revealing a postreceptoral target for NO-induced modulation of [Ca²⁺] mobilization. Using a lys⁹-biotinylated ET-1 (ET-1 [BtK⁹]), binding sites were “mapped” in CHO-ET cells. Receptor-ligand complexes did not exhibit spontaneous dissociation during 60min observations. Quantitative fluorescence microscopy revealed that SNP or SIN-1 caused a rapid, concentration-dependent, and reversible dissociation of biotinylated ET- 1 from ET_A receptor (EC₅₀ = 75 μM and 6 μM, respectively), an effect that was not mimicked by 8-bromo-cyclic GMP. “Sandwich” co-culture of endothelial cells with CHO-ET showed that activation of NO production by endothelial cells similarly resulted in dissociation of ET-1 [BtK⁹] from ET_A receptors. We hypothesize that NO plays a role in physiological termination of ET-1 signalling by dual mechanisms: (1) displacement of bound ET-1 from its receptor, thus preventing homologous desensitization, and (2) interference with the postreceptoral pathway for [Ca²⁺] mobilization, hence inhibiting end-responses to ET-1. © 1994 Wiley-Liss, Inc.     

(2S)-2-Amino-5-chloro-4-hydroxy-5-hexenoic acid,a new chloroamino acid,and related compounds fromAmanita gymnopus

Combining different chromatography systems, unusual nonprotein amino acids were isolated and unequivocally identified from a small amount (less than 100 g fresh weight) ofAmanita gymnopus fruit body. Without obtaining crystals of these amino acids, on the basis of¹H-NMR determination, high resolution mass spectrometry, chlorine analysis and oxidation with L-amino acid oxidase, one of them proved to be a new chloroamino acid, (2S)-2-amino-5-chloro-4-hydroxy-5-hexenoic acid (G2). The other three were (2S)-2-amino-5-hexenoic acid (G1), (2S)-2-amino-4,5-hexadienoic acid (G3) and (2S)-2-amino-5-hexynoic acid (G4). Amino acid (G1) was also encountered for the first time in natural products. Amino acid (G3) has been reported from several kinds of fungi belonging toAmanita, subgenusLepidella. The occurrence of amino acid (G4) was already reported fromCortinarius claricolor.Part 23 in the series Biochemical studies of nitrogen compounds in fungi. Part 22, Hatanaka, S. I. et al. 1985. Trans. Mycol. Soc. Japan26: 61–68.     

Integrated tandem affinity protein purification using the polyhistidine plus extra 4 amino acids (HiP4) tag system

Peptide tag systems are a robust biophysical and biochemical method that is widely used for protein detection and purification. Here, we developed a novel tag system termed “HiP4” (histidine plus four amino acids) whose epitope sequence comprises only seven amino acids (HHHDYDI) that partially overlap with the conventional 6x histidine tag (6xHis-tag). We produced a monoclonal antibody against the HiP4 tag that can be used in multiple immunoassays with high specificity and affinity. Using this system, we developed a tandem affinity purification (TAP) and mass spectrometry (TAP-MS) system for comprehensive protein interactome analysis. The integrated use of nickel bead purification followed by HiP4 tag immunoprecipitation made it possible to reduce nonspecific binding and improve selectivity, leading to the recovery of previously unrecognized proteins that interact with hepatitis B virus X (HBx) protein or TAR DNA-binding protein 43 (TARDBP or TDP-43). Our results indicate that this system may be viable as a simple and powerful tool for TAP-MS that can achieve low background and high selectivity in comprehensive protein–protein interaction analyses.     

Molecular cloning of two tandemly arranged peroxidase genes from Populus kitakamiensis and their differential regulation in the stem

A genomic library was prepared from Populus kitakamiensis and screened with the cDNA for an anionic peroxidase from P. kitakamiensis. One genomic clone was isolated that contained two tandemly oriented genes for anionic peroxidases, prxA3a and prxA4a. Both genes consisted of four exons and three introns; the introns had consensus nucleotides, namely, GT and AG, at their 5 and 3 ends, respectively. The prxA3a and prxA4a genes encoded 347 and 343 amino acid residues, respectively, including putative signal sequences at the amino-termini. Putative promoters and polyadenylation signals were found in the flanking regions of both genes. The sequence of the coding region of prxA3a was completely identical to that of the cDNA clone pA3, whereas the sequence of the coding region of prxA4a was only 73% identical to that of the cDNA clone pA3. Northern blot analysis showed that the patterns of expression of the mRNAs that corresponded to prxA3a and prxA4a differed in stems of P. kitakamiensis.