scholarly journals Chromosomal Location Plays a Role in Regulation of Aflatoxin Gene Expression in Aspergillus parasiticus

2002 ◽  
Vol 68 (1) ◽  
pp. 306-315 ◽  
Author(s):  
Ching-Hsun Chiou ◽  
Michael Miller ◽  
David L. Wilson ◽  
Frances Trail ◽  
John E. Linz
Keyword(s):  
Gene Cluster ◽  
Promoter Activity ◽  
Aspergillus Parasiticus ◽  
Chromosomal Location ◽  
Selectable Marker ◽  
Growth Conditions ◽  
Gus Expression ◽  
Reporter System ◽  
Gus Reporter ◽  
Homologous Site

ABSTRACT The nor-1 gene in the filamentous fungus Aspergillus parasiticus encodes a ketoreductase involved in aflatoxin biosynthesis. To study environmental influences on nor-1 expression, we generated plasmid pAPGUSNNB containing a nor-1 promoter-β-glucuronidase (GUS) (encoded by uidA) reporter fusion with niaD (encodes nitrate reductase) as a selectable marker. niaD transformants of A. parasiticus strain NR-1 (niaD) carried pAPGUSNNB integrated predominantly at the nor-1 or niaD locus. Expression of the native nor-1 and nor-1::GUS reporter was compared in transformants grown under aflatoxin-inducing conditions by Northern and Western analyses and by qualitative and quantitative GUS activity assays. The timing and level of nor-1 promoter function with pAPGUSNNB integrated at nor-1 was similar to that observed for the native nor-1 gene. In contrast, nor-1 promoter activity in pAPGUSNNB and a second nor-1::GUS reporter construct, pBNG3.0, was not detectable when integration occurred at niaD. Because niaD-dependent regulation could account for the absence of expression at niaD, a third chromosomal location was analyzed using pAPGUSNP, which contained nor-1::GUS plus pyrG (encodes OMP decarboxylase) as a selectable marker. GUS expression was detectable only when pAPGUSNP integrated at nor-1 and was not detectable at pyrG, even under growth conditions that required pyrG expression. nor-1::GUS is regulated similarly to the native nor-1 gene when it is integrated at its homologous site within the aflatoxin gene cluster but is not expressed at native nor-1 levels at two locations outside of the aflatoxin gene cluster. We conclude that the GUS reporter system can be used effectively to measure nor-1 promoter activity and that nor-1 is subject to position-dependent regulation in the A. parasiticus chromosome.

Production of Aflatoxin in Cocoa Beans

1979 ◽  
Vol 62 (5) ◽  
pp. 1076-1079 ◽  
Author(s):  
Lawrence M Lenovich ◽  
W Jeffrey Hurst
Keyword(s):  
High Pressure ◽  
Liquid Chromatography ◽  
Ivory Coast ◽  
Aspergillus Parasiticus ◽  
Aflatoxin Production ◽  
Growth Conditions ◽  
Aflatoxin Contamination ◽  
Cocoa Beans ◽  
Total Aflatoxin ◽  
Substrate Variation

Abstract Aflatoxin was produced in both non-autoclaved and autoclaved Ivory Coast cocoa beans inoculated with Aspergillus parasiticus NRRL 2999 under optimum laboratory growth conditions. Total aflatoxin levels ranged from 213 to 5597 ng/g substrate. Aflatoxin was quantitated by using high pressure liquid chromatography (HPLC). Raw, non-autoclaved cocoa beans, also inoculated with aspergilli, produced 6359 ng aflatoxin/g substrate. Variation in aflatoxin production between bean varieties was observed. Total aflatoxin levels of 10,446 and 23,076 ng/g substrate were obtained on Ivory Coast beans inoculated with A. parasiticus NRRL 2999 and NRRL 3240, respectively. Aflatoxin production on Trinidad and Malaysian beans was 28 and 65 ng aflatoxin/g substrate. These data support previously reported low level natural aflatoxin contamination in cocoa.

scholarly journals Characterization of two cis-acting elements, P1BS and W-box, in the regulation of OsPT6 responsive to phosphors deficiency

2021 ◽  
Author(s):  
Qingchun Zhao ◽  
Zhenzhen Luo ◽  
Jiadong Chen ◽  
Hongfang Jia ◽  
Penghui Ai ◽  
...  
Keyword(s):  
Gus Expression ◽  
Element Analysis ◽  
P Deficiency ◽  
Targeted Deletion ◽  
Cis Acting ◽  
Gus Reporter ◽  
Pi Starvation ◽  
Expression Activity ◽  
Underlying Mechanisms ◽  
Diverse Plant Species

AbstractPhosphorus (P) deficiency is one of the major nutrient stresses restricting plant growth. The uptake of P by plants from soil is mainly mediated by the phosphate (Pi) transporters belonging to the PHT1 family. Multiple PHT1 genes from diverse plant species have been shown to be strongly up-regulated upon Pi starvation, however, the underlying mechanisms for the Pi-starvation-induced (PSI) up-regulation have not been well deciphered for most Pi transporter genes. Here, we reported a detailed dissection of the promoter activity of a PSI rice Pi transporter gene OsPT6, using the β-glucuronidase (GUS) reporter gene. OsPT6 promoter could drive GUS expression strongly in both roots and blades of rice plants grown under low P, but not high P. Cis-acting element analysis identified one copy of the P1BS motif and two copies of the W-box motif in OsPT6 promoter. Targeted deletion of the P1BS motif caused almost complete abolition of GUS induction in response to Pi starvation, irrespective of the presence or absence of the W-box motif, Four repeats of the P1BS motif fused to the CaMV35S minimal promoter was sufficient to induce GUS expression responsive to Pi starvation. Targeted deletion of the upstream W-box motif (W1) did not affect the GUS expression activity compared with the full-length OsPT6 promoter, while targeted deletion of the downstream W-box motif (W2) or both of the W-box motifs remarkably reduced the GUS induction rate upon Pi starvation. Our results proposed that the PSI response of OsPT6 was positively regulated by at least two elements, the sole P1BS and the downstream W-box, in its promoter, and the W-box-mediated up-regulation of OsPT6 might be highly dependent on the P1BS motif.

scholarly journals Bioactivity guided isolation of phytoestrogenic compounds from Cyclopia genistoides by the pER8:GUS reporter system

2017 ◽  
Vol 110 ◽  
pp. 201-207 ◽  
Author(s):  
O. Roza ◽  
W.-C. Lai ◽  
I. Zupkó ◽  
J. Hohmann ◽  
N. Jedlinszki ◽  
...  
Keyword(s):  
Reporter System ◽  
Gus Reporter ◽  
Gus Reporter System

scholarly journals Overexpression of a Redox-Regulated Cutinase Gene, MfCUT1, Increases Virulence of the Brown Rot Pathogen Monilinia fructicola on Prunus spp.

2010 ◽  
Vol 23 (2) ◽  
pp. 176-186 ◽  
Author(s):  
Miin-Huey Lee ◽  
Chiu-Min Chiu ◽  
Tatiana Roubtsova ◽  
Chien-Ming Chou ◽  
Richard M. Bostock
Keyword(s):  
Gene Expression ◽  
Redox Regulation ◽  
Brown Rot ◽  
Gus Expression ◽  
Apple Fruit ◽  
Gus Activity ◽  
Monilinia Fructicola ◽  
Gus Reporter ◽  
Flanking Regions ◽  
Prunus Spp

A 4.5-kb genomic DNA containing a Monilinia fructicola cutinase gene, MfCUT1, and its flanking regions were isolated and characterized. Sequence analysis revealed that the genomic MfCUT1 carries a 63-bp intron and a promoter region with several transcription factor binding sites that may confer redox regulation of MfCUT1 expression. Redox regulation is indicated by the effect of antioxidants, shown previously to inhibit MfCUT1 gene expression in cutin-induced cultures, and in the present study, where H2O2 enhanced MfCUT1 gene expression. A β-glucuronidase (GUS) reporter gene (gusA) was fused to MfCUT1 under the control of the MfCUT1 promoter, and this construct was then used to generate an MfCUT1-GUS strain by Agrobacterium spp.-mediated transformation. The appearance of GUS activity in response to cutin and suppression of GUS activity by glucose in cutinase-inducing medium verified that the MfCUT1-GUS fusion protein was expressed correctly under the control of the MfCUT1 promoter. MfCUT1-GUS expression was detected following inoculation of peach and apple fruit, peach flower petals, and onion epidermis, and during brown rot symptom development on nectarine fruit at a relatively late stage of infection (24 h postinoculation). However, semiquantitative reverse-transcriptase polymerase chain reaction provided sensitive detection of MfCUT1 expression within 5 h of inoculation in both almond and peach petals. MfCUT1-GUS transformants expressed MfCUT1 transcripts at twice the level as the wild type and caused more severe symptoms on Prunus flower petals, consistent with MfCUT1 contributing to the virulence of M. fructicola.

scholarly journals Molecular Characterization of the Leucine Cluster in Buchnera sp. Strain PSY, a Primary Endosymbiont of the Aphid Pemphigus spyrothecae

2002 ◽  
Vol 68 (5) ◽  
pp. 2572-2575 ◽  
Author(s):  
Beatriz Sabater-Muñoz ◽  
Laura Gómez-Valero ◽  
Roeland C. H. J. van Ham ◽  
Francisco J. Silva ◽  
Amparo Latorre
Keyword(s):  
Gene Cluster ◽  
Molecular Characterization ◽  
Chromosomal Location ◽  
Pemphigus Spyrothecae

ABSTRACT Buchnera strains from most aphid subfamilies studied to date have been found to carry the leucine gene cluster (leuA, -B, -C, and -D) on a plasmid, an organization unique among bacteria. Here, however, we demonstrate a classical chromosomal location of the cluster in Buchnera sp. strain PSY from the aphid Pemphigus spyrothecae (subfamily Pemphiginae). The genes that flank leuABCD in Buchnera sp. strain PSY appear to be adjacent in the genome of Buchnera sp. strain APS, a strain carrying a leucine plasmid. We propose that the presence of a leucine plasmid predates the diversification of symbiotic Buchnera and that the chromosomal location observed in Buchnera sp. strain PSY arose by a transfer of the leucine genes from a plasmid to the chromosome.

scholarly journals Characteristics of the promoters derived from the single-stranded DNA components of Milk vetch dwarf virus in transgenic tobacco

2005 ◽  
Vol 86 (6) ◽  
pp. 1851-1860 ◽  
Author(s):  
Naomi Shirasawa-Seo ◽  
Yoshitaka Sano ◽  
Shigeo Nakamura ◽  
Taka Murakami ◽  
Shigemi Seo ◽  
...  
Keyword(s):  
Transgenic Tobacco ◽  
Cell Types ◽  
Gus Expression ◽  
Cauliflower Mosaic Virus ◽  
Dwarf Virus ◽  
Promoter Regions ◽  
Meristematic Tissue ◽  
Link Protein ◽  
Gus Reporter ◽  
Milk Vetch Dwarf Virus

Predicted promoter regions of Milk vetch dwarf virus (MDV) components (C1–C11) were isolated and fused with a β-glucuronidase (GUS) reporter gene and the characteristics of the promoters were examined. In transgenic tobacco calli, promoters of MDV C4 (encoding a cell-cycle link protein), C5 and C7 (both encoding unknown proteins), C6 (encoding a nuclear-shuttle protein) and C8 (encoding a movement protein) generated a stronger level of GUS expression than the Cauliflower mosaic virus 35S RNA promoter (P35S). In leaves of transgenic tobacco plants, the promoters of C5 and C8 conferred a level of GUS activity comparable to that of P35S. Histochemical GUS analysis showed that the promoters of C4–C9, the latter encoding a capsid protein, were active in phloem and meristematic tissue. The promoter of C8 was also active in mesophyll and cortex cell types. A low level of activity was found for the promoters of C11, which encodes a master replication-initiator protein (Rep), and C1, C2, C3 and C10, which encode additional Reps, in both transgenic tobacco calli and plants.

scholarly journals Identification of the Core Pollen-Specific Regulation in the Rice OsSUT3 Promoter

2020 ◽  
Vol 21 (6) ◽  
pp. 1909
Author(s):  
Dandan Li ◽  
Rucong Xu ◽  
Dong Lv ◽  
Chunlong Zhang ◽  
Hong Yang ◽  
...  
Keyword(s):  
Pollen Development ◽  
Ectopic Expression ◽  
Gus Expression ◽  
35S Promoter ◽  
Crop Breeding ◽  
Specific Expression ◽  
Regulatory Motifs ◽  
Gus Reporter ◽  
Specific Regulation ◽  
Promoter Deletions

The regulatory mechanisms of pollen development have potential value for applications in agriculture, such as better understanding plant reproductive regularity. Pollen-specific promoters are of vital importance for the ectopic expression of functional genes associated with pollen development in plants. However, there is a limited number of successful applications using pollen-specific promoters in genetic engineering for crop breeding and hybrid generation. Our previous work led to the identification and isolation of the OsSUT3 promoter from rice. In this study, to analyze the effects of different putative regulatory motifs in the OsSUT3 promoter, a series of promoter deletions were fused to a GUS reporter gene and then stably introduced into rice and Arabidopsis. Histochemical GUS analysis of transgenic plants revealed that p385 (from −385 to −1) specifically mediated maximal GUS expression in pollen tissues. The S region (from −385 to −203) was the key region for controlling the pollen-specific expression of a downstream gene. The E1 (−967 to −606), E2 (−202 to −120), and E3 (−119 to −1) regions enhanced ectopic promoter activity to different degrees. Moreover, the p385 promoter could alter the expression pattern of the 35S promoter and improve its activity when they were fused together. In summary, the p385 promoter, a short and high-activity promoter, can function to drive pollen-specific expression of transgenes in monocotyledon and dicotyledon transformation experiments.

scholarly journals Unraveling the regulation of sophorolipid biosynthesis in Starmerella bombicola

2020 ◽  
Vol 20 (3) ◽  
Author(s):  
Sofie Lodens ◽  
Sophie L K W Roelants ◽  
Goedele Luyten ◽  
Robin Geys ◽  
Pieter Coussement ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stationary Phase ◽  
Gene Cluster ◽  
Biosynthetic Gene Cluster ◽  
Exponential Phase ◽  
Biosynthetic Gene ◽  
Reporter System ◽  
Gfp Expression ◽  
Qpcr Analysis ◽  
Starmerella Bombicola

ABSTRACT Starmerella bombicola very efficiently produces the secondary metabolites sophorolipids (SLs). Their biosynthesis is not-growth associated and highly upregulated in the stationary phase. Despite high industrial and academic interest, the underlying regulation of SL biosynthesis remains unknown. In this paper, potential regulation of SL biosynthesis through the telomere positioning effect (TPE) was investigated, as the SL gene cluster is located adjacent to a telomere. An additional copy of this gene cluster was introduced elsewhere in the genome to investigate if this results in a decoy of regulation. Indeed, for the new strain, the onset of SL production was shifted to the exponential phase. This result was confirmed by RT-qPCR analysis. The TPE effect was further investigated by developing and applying a suitable reporter system for this non-conventional yeast, enabling non-biased comparison of gene expression between the subtelomeric CYP52M1- and the URA3 locus. This was done with a constitutive endogenous promotor (pGAPD) and one of the endogenous promotors of the SL biosynthetic gene cluster (pCYP52M1). A clear positioning effect was observed for both promotors with significantly higher GFP expression levels at the URA3 locus. No clear GFP upregulation was observed in the stationary phase for any of the new strains.

scholarly journals Function of the cypX and moxY Genes in Aflatoxin Biosynthesis in Aspergillus parasiticus

2005 ◽  
Vol 71 (6) ◽  
pp. 3192-3198 ◽  
Author(s):  
Ying Wen ◽  
Hidemi Hatabayashi ◽  
Hatsue Arai ◽  
Hiroko K. Kitamoto ◽  
Kimiko Yabe
Keyword(s):  
Gene Cluster ◽  
Rapid Method ◽  
Aspergillus Parasiticus ◽  
Cell Disruption ◽  
Aflatoxin Biosynthesis ◽  
Feeding Experiments ◽  
Phenol Extraction ◽  
Versiconal Hemiacetal Acetate

ABSTRACT The pathway oxoaverantin (OAVN) → averufin (AVR) → hydroxyversicolorone (HVN) → versiconal hemiacetal acetate (VHA) is involved in aflatoxin biosynthesis, and the cypX and moxY genes, which are present in the aflatoxin gene cluster, have been previously suggested to be involved in this pathway. To clarify the function of these two genes in more detail, we disrupted the genes in aflatoxigenic Aspergillus parasiticus NRRL 2999. The cypX-deleted mutant lost aflatoxin productivity and accumulated AVR in the mycelia. Although this mutant converted HVN, versicolorone (VONE), VHA, and versiconol acetate (VOAc) to aflatoxins in feeding experiments, it could not produce aflatoxins from either OAVN or AVR. The moxY-deleted mutant also lost aflatoxin productivity, whereas it newly accumulated HVN and VONE. In feeding experiments, this mutant converted either VHA or VOAc to aflatoxins but did not convert OAVN, AVR, HVN, or VONE to aflatoxins. These results demonstrated that cypX encodes AVR monooxygenase, catalyzing the reaction from AVR to HVN, and moxY encodes HVN monooxygenase, catalyzing a Baeyer-Villiger reaction from HVN to VHA as well as from VONE to VOAc. In this work, we devised a simple and rapid method to extract DNA from many fungi for PCR analyses in which cell disruption with a shaker and phenol extraction were combined.

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