scholarly journals Construction of a Codon-Adapted Nourseotricin-Resistance Marker Gene for Efficient Targeted Gene Deletion in the Mycophenolic Acid Producer Penicillium brevicompactum

2019 ◽  
Vol 5 (4) ◽  
pp. 96
Author(s):  
Yasaman Mahmoudjanlou ◽  
Birgit Hoff ◽  
Ulrich Kück
Keyword(s):  
Mycophenolic Acid ◽  
Marker Gene ◽  
Wild Type ◽  
Resistance Marker ◽  
Subsequent Transformation ◽  
Targeted Gene Deletion ◽  
Filamentous Ascomycete ◽  
Wild Type Phenotype ◽  
Penicillium Brevicompactum ◽  
Resistance Markers

Penicillium brevicompactum is a filamentous ascomycete used in the pharmaceutical industry to produce mycophenolic acid, an immunosuppressant agent. To extend options for genetic engineering of this fungus, we have tested two resistance markers that have not previously been applied to P. brevicompactum. Although a generally available phleomycin resistance marker (ble) was successfully used in DNA-mediated transformation experiments, we were not able to use a commonly applicable nourseothricin resistance cassette (nat1). To circumvent this failure, we constructed a new nat gene, considering the codon bias for P. brevicompactum. We then used this modified nat gene in subsequent transformation experiments for the targeted disruption of two nuclear genes, MAT1-2-1 and flbA. For MAT1-2-1, we obtained deletion strains with a frequency of about 10%. In the case of flbA, the frequency was about 4%, and this disruption strain also showed reduced conidiospore formation. To confirm the deletion, we used ble to reintroduce the wild-type genes. This step restored the wild-type phenotype in the flbA deletion strain, which had a sporulation defect. The successful transformation system described here substantially extends options for genetically manipulating the biotechnologically relevant fungus P. brevicompactum.

scholarly journals The mechanism of phenylpyrrole fungicides cannot be explained by a stearic effect upon triososephosphate isomerase alone

2020 ◽  
Author(s):  
Stefan Jacob ◽  
T. Tristan Brandhorst
Keyword(s):  
Drug Target ◽  
Triosephosphate Isomerase ◽  
Marker Gene ◽  
Physical Interaction ◽  
Resistance Marker ◽  
Filamentous Ascomycete ◽  
Direct Target ◽  
And Oxidative Stress ◽  
Null Mutants

In a Scientific Reports article by Brandhorst et al. it was demonstrated that fludioxonil elicited aldehydic and oxidative stress, and that aldehydic stress could mimic the effects of fludioxonil upon fludioxonil sensitive fungi. As triosephosphate isomerase (TPI) can evolve aldehydic stress in the form of methylglyoxal (MGO), and in vitro evidence showed that fludioxonil lead to an elevation of the release of this MGO, it was postulated that TPI may be a target of fludioxonil. Unfortunately, some have interpreted this article to imply that TPI is the sole direct drug target of this fungicide class. This conclusion is not fully supported by the data, however, as experimental evidence of a direct physical interaction between fludioxonil and TPI in the pathogen has been lacking. Now, further investigation by the Jacob group clearly demonstrates that TPI cannot be the sole direct target of fludioxonil action. We have specifically inactivated the coding sequence of TPI in the genome of the phytopathogenic filamentous ascomycete Magnaporthe oryzae (anamorph: Pyricularia oryzae). We replaced it with a resistance marker gene and proved the successful deletion of the gene. Were the TPI enzyme the sole direct target of the phenylpyrrole fludioxonil, the null mutants ΔMotpi should be resistant to fludioxonil compared to the wildtype strain upon phenylpyrrole treatment. This is not the case.

scholarly journals In Vitro Combination of Amdoxovir and the Inosine Monophosphate Dehydrogenase Inhibitors Mycophenolic Acid and Ribavirin Demonstrates Potent Activity against Wild-Type and Drug-Resistant Variants of Human Immunodeficiency Virus Type 1

2004 ◽  
Vol 48 (11) ◽  
pp. 4387-4394 ◽  
Author(s):  
Katyna Borroto-Esoda ◽  
Florence Myrick ◽  
Joy Feng ◽  
Jerry Jeffrey ◽  
Phillip Furman
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Mycophenolic Acid ◽  
Inosine Monophosphate Dehydrogenase ◽  
Wild Type ◽  
Inosine Monophosphate ◽  
Immunodeficiency Virus ◽  
Monophosphate Dehydrogenase

ABSTRACT Amdoxovir [(−)-β-d-2,6-diaminopurine dioxolane (DAPD)] is a nucleoside analogue reverse transcriptase inhibitor of human immunodeficiency virus type 1 (HIV-1) replication. DAPD is deaminated by adenosine deaminase to the guanosine analogue dioxolane guanosine (DXG), which is subsequently phosphorylated to the corresponding 5′ triphosphate (DXG-TP). DXG-TP competes with the natural substrate dGTP for binding to the enzyme-nucleic acid complex. Mycophenolic acid (MPA) and ribavirin (RBV), inhibitors of inosine monophosphate dehydrogenase (IMPDH), inhibit the de novo synthesis of guanine nucleotides, including dGTP. Reducing the intracellular levels of dGTP would be expected to augment the antiviral activity of analogues of deoxyguanosine. In this study we examined the effect of MPA and RBV on the anti-HIV activity of DAPD and DXG. When tested against wild-type virus, both MPA and RBV decreased the 50% effective concentration (EC50) for DXG by at least 10-fold. In contrast, both MPA and RBV increase the EC50 value for zidovudine. MPA and RBV completely reversed the resistance to DXG observed with HIV isolates containing mutations which confer partial resistance to DAPD and DXG. Similarly, when tested against a mutant virus fully resistant to inhibition by DAPD (K65R/Q151M), MPA and RBV reduced the EC50 for DAPD to within twofold of that for the wild type. The combination of MPA or RBV with DAPD or DXG did not result in increased cytotoxicity or reduced levels of mitochondrial DNA when tested at physiologically relevant concentrations. These studies suggest a potential role for the use of IMPDH inhibitors in combination therapy with amdoxovir in the treatment of HIV.

scholarly journals The UDP N-Acetylgalactosamine 4-Epimerase Gene Is Essential for Mesophilic Aeromonas hydrophila Serotype O34 Virulence

2006 ◽  
Vol 74 (1) ◽  
pp. 537-548 ◽  
Author(s):  
Rocío Canals ◽  
Natalia Jiménez ◽  
Silvia Vilches ◽  
Miguel Regué ◽  
Susana Merino ◽  
...  
Keyword(s):  
Aeromonas Hydrophila ◽  
Type Strain ◽  
Wild Type Strain ◽  
Sugar Residue ◽  
Serum Resistance ◽  
Single Mutation ◽  
Wild Type ◽  
O Antigen ◽  
Wild Type Phenotype

ABSTRACT Mesophilic Aeromonas hydrophila strains of serotype O34 typically express smooth lipopolysaccharide (LPS) on their surface. A single mutation in the gene that codes for UDP N-acetylgalactosamine 4-epimerase (gne) confers the O− phenotype (LPS without O-antigen molecules) on a strain in serotypes O18 and O34, but not in serotypes O1 and O2. The gne gene is present in all the mesophilic Aeromonas strains tested. No changes were observed for the LPS core in a gne mutant from A. hydrophila strain AH-3 (serotype O34). O34 antigen LPS contains N-acetylgalactosamine, while no such sugar residue forms part of the LPS core from A. hydrophila AH-3. Some of the pathogenic features of A. hydrophila AH-3 gne mutants are drastically reduced (serum resistance or adhesion to Hep-2 cells), and the gne mutants are less virulent for fish and mice compared to the wild-type strain. Strain AH-3, like other mesophilic Aeromonas strains, possess two kinds of flagella, and the absence of O34 antigen molecules by gne mutation in this strain reduced motility without any effect on the biogenesis of both polar and lateral flagella. The reintroduction of the single wild-type gne gene in the corresponding mutants completely restored the wild-type phenotype (presence of smooth LPS) independently of the O wild-type serotype, restored the virulence of the wild-type strain, and restored motility (either swimming or swarming).

Rescue of the mutant phenotype by reexpression of full-length vinculin in null F9 cells; effects on cell locomotion by domain deleted vinculin

1998 ◽  
Vol 111 (11) ◽  
pp. 1535-1544 ◽  
Author(s):  
W. Xu ◽  
J.L. Coll ◽  
E.D. Adamson
Keyword(s):  
Marked Effect ◽  
Control Cell ◽  
Covalent Attachment ◽  
Tail Domain ◽  
Wild Type ◽  
F9 Cells ◽  
Wild Type Phenotype ◽  
Correct Location ◽  
Low Levels ◽  
Lamellipodia Formation

Vinculin plays a role in signaling between integrins and the actin cytoskeleton. We reported earlier that F9-derived cells lacking vinculin are less spread, less adhesive, and move two times faster than wild-type F9 cells. Expression of intact vinculin in null cells restored all wild-type characteristics. In contrast, expression of the head (90 kDa) fragment exaggerated mutant characteristics, especially locomotion, which was double that of vinculin null cells. Expression of the tail domain also had a marked effect on locomotion in the opposite direction, reducing it to very low levels. The expression of the head plus tail domains together (no covalent attachment) effected a partial rescue towards wild-type phenotype, thus indicating that reexpressed polypeptides may be in their correct location and are interacting normally. Therefore, we conclude that: (1) the head domain is part of the locomotory force of the cell, modulated by the tail, and driven by the integrin/matrix connection; (2) intact vinculin is required for normal regulation of cell behavior, suggesting that vinculin head-tail interactions control cell adhesion, spreading, lamellipodia formation and locomotion.

scholarly journals New Sporulation Loci in Streptomyces coelicolor A3(2)

1999 ◽  
Vol 181 (17) ◽  
pp. 5419-5425 ◽  
Author(s):  
N. Jamie Ryding ◽  
Maureen J. Bibb ◽  
Virginie Molle ◽  
Kim C. Findlay ◽  
Keith F. Chater ◽  
...  
Keyword(s):  
Phase Contrast ◽  
Streptomyces Coelicolor ◽  
Cosmid Library ◽  
Phase Contrast Microscopy ◽  
Wild Type ◽  
Chromosomal Dna ◽  
East Anglia ◽  
Wild Type Phenotype ◽  
Contrast Microscopy ◽  
Spore Pigment

ABSTRACT Sporulation mutants of Streptomyces coelicolor appear white because they are defective in the synthesis of the grey polyketide spore pigment, and such white (whi) mutants had been used to define eight sporulation loci, whiA,whiB, whiD, whiE, whiG,whiH, whiI, and whiJ (K. F. Chater, J. Gen. Microbiol. 72:9–28, 1972; N. J. Ryding, Ph.D. thesis, University of East Anglia, 1995). In an attempt to identify new whi loci, we mutagenized S. coelicolor M145 spores with nitrosoguanidine and identified 770 mutants with colonies ranging from white to medium grey. After excluding unstable strains, we examined the isolates by phase-contrast microscopy and chose 115 whi mutants with clear morphological phenotypes for further study. To exclude mutants representing cloned whi genes, self-transmissible SCP2*-derived plasmids carrying whiA, whiB,whiG, whiH, or whiJ (but notwhiD, whiE, or whiI) were introduced into each mutant by conjugation, and strains in which the wild-type phenotype was restored either partially or completely by any of these plasmids were excluded from further analysis. In an attempt to complement some of the remaining 31 whi mutants, an SCP2* library of wild-type S. coelicolor chromosomal DNA was introduced into 19 of the mutants by conjugation. Clones restoring the wild-type phenotype to 12 of the 19 strains were isolated and found to represent five distinct loci, designated whiK,whiL, whiM, whiN, andwhiO. Each of the five loci was located on the ordered cosmid library: whiL, whiM, whiN, and whiO occupied positions distinct from previously clonedwhi genes; whiK was located on the same cosmid overlap as whiD, but the two loci were shown by complementation to be distinct. The phenotypes resulting from mutations at each of these new loci are described.

Mycophenolic acid is produced during balanced growth of Penicillium brevicompactum

1977 ◽  
Vol 23 (1) ◽  
pp. 20-27 ◽  
Author(s):  
Carter P. Nulton ◽  
Iain M. Campbell
Keyword(s):  
Dilution Rate ◽  
Secondary Metabolite ◽  
Mycophenolic Acid ◽  
Continuous Flow ◽  
Acid Production ◽  
Flow System ◽  
Metabolic System ◽  
Balanced Growth ◽  
Secondary Metabolite Biosynthesis ◽  
Penicillium Brevicompactum

When Penicillium brevicompactum is grown on Czapek Dox medium in the surface or sub merged mode as batch or continuous-flow cultures, mycophenolic acid is produced. Unlike the classical secondary metabolic system, 6-methylsalicylic acid production by P. patulum, mycophenolic acid is formed independently of dilution rate in a flow system. Discounting the possibility that strains of P. brevicompactum that produce mycophenolic acid are mutants defective in the control of secondary metabolite biosynthesis, we conclude that mycophenolic acid production is not regulated as part of a non-vegetative genome. An invertase (EC 3.2.1.26) activity has been encountered in both P. brevicompactum and P. patulum.

scholarly journals Association of intronic DNA methylation and hydroxymethylation alterations in the epigenetic etiology of dilated cardiomyopathy

2019 ◽  
Vol 317 (1) ◽  
pp. H168-H180 ◽  
Author(s):  
Ali M. Tabish ◽  
Mohammed Arif ◽  
Taejeong Song ◽  
Zaher Elbeck ◽  
Richard C. Becker ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Dilated Cardiomyopathy ◽  
Cardiac Remodeling ◽  
Rna Seq ◽  
Wild Type ◽  
Kegg Pathways ◽  
Wild Type Phenotype ◽  
Gene Expression Levels

In this study, we investigated the role of DNA methylation [5-methylcytosine (5mC)] and 5-hydroxymethylcytosine (5hmC), epigenetic modifications that regulate gene activity, in dilated cardiomyopathy (DCM). A MYBPC3 mutant mouse model of DCM was compared with wild type and used to profile genomic 5mC and 5hmC changes by Chip-seq, and gene expression levels were analyzed by RNA-seq. Both 5mC-altered genes (957) and 5hmC-altered genes (2,022) were identified in DCM hearts. Diverse gene ontology and KEGG pathways were enriched for DCM phenotypes, such as inflammation, tissue fibrosis, cell death, cardiac remodeling, cardiomyocyte growth, and differentiation, as well as sarcomere structure. Hierarchical clustering of mapped genes affected by 5mC and 5hmC clearly differentiated DCM from wild-type phenotype. Based on these data, we propose that genomewide 5mC and 5hmC contents may play a major role in DCM pathogenesis. NEW & NOTEWORTHY Our data demonstrate that development of dilated cardiomyopathy in mice is associated with significant epigenetic changes, specifically in intronic regions, which, when combined with gene expression profiling data, highlight key signaling pathways involved in pathological cardiac remodeling and heart contractile dysfunction.

scholarly journals Interactions Between Antenatal Sulfadoxine-Pyrimethamine, Drug-Resistant Plasmodium falciparum Parasites, and Delivery Outcomes in Malawi

2020 ◽  
Vol 222 (4) ◽  
pp. 661-669 ◽  
Author(s):  
Steve M Taylor ◽  
Brandt Levitt ◽  
Betsy Freedman ◽  
Mwayiwawo Madanitsa ◽  
Kyaw-Lay Thwai ◽  
...  
Keyword(s):  
Birth Weight ◽  
Preventive Therapy ◽  
Sub Saharan Africa ◽  
Drug Resistant ◽  
Wild Type ◽  
Resistance Marker ◽  
Chain Reaction ◽  
Delivery Outcomes ◽  
Polymerase Chain ◽  
Sub Saharan

Abstract Background Sulfadoxine-pyrimethamine (SP) is used as intermittent preventive therapy in pregnancy (IPTp) for malaria in sub-Saharan Africa. The resistance marker dhps A581G has been associated with reduced IPTp-SP efficacy and enhanced morbidity in SP recipients. Methods We measured SP-resistance allele frequencies in Malawian women participating in a trial comparing IPTp with SP against intermittent screening by rapid diagnostic tests (ISTp). We genotyped polymerase chain reaction-detected parasites using deep sequencing of SP-resistance alleles. Results Among 125 placental infections, A581G-bearing parasites were associated with reduced birth weight (mean difference [MD], 252 g; 95% confidence interval [CI], 46–457; P = .017). Relative to ISTp, IPTp-SP was associated with higher birth weights in women with wild-type parasites (MD, 116 g; 95% CI, −40 to 272; P = .142) and lower birth weights in women with A581G-bearing parasites (MD, 192 g; 95% CI, −264 to 648; P = .385) (Pinteraction = .033). Similar associations were noted on gestational age (Pinteraction = .075). Amongst only IPTp-SP recipients, relative to women who last received SP > 4 weeks before delivery, recent SP receipt was associated with lower birth weight in women with wild-type parasites (MD, 118 g; 95% CI, −376 to 139; P = .361) and higher birth weight in women with A581G-bearing parasites (MD, 783 g; 95% CI, −20 to 1586; P = .054) (Pinteraction = .005). Conclusions The effectiveness in birth weight of IPTp-SP is compromised by A581G-bearing parasites, but there was no evidence that the adverse effects of these parasites are exacerbated by antenatal SP. ISRCTN Registry www.isrctn.com/ISRCTN69800930.

scholarly journals Micro-RNA Clusters Integrate Evolutionary Constraints on Expression and Target Affinities: The miR-6/5/4/286/3/309 Cluster in Drosophila

2020 ◽  
Vol 37 (10) ◽  
pp. 2955-2965
Author(s):  
Qu Zhe ◽  
Wing Chung Yiu ◽  
Ho Yin Yip ◽  
Wenyan Nong ◽  
Clare W C Yu ◽  
...  
Keyword(s):  
Micro Rna ◽  
Small Rna Sequencing ◽  
Wild Type ◽  
New Members ◽  
Cluster Evolution ◽  
Rna Targets ◽  
Wild Type Phenotype ◽  
Micro Rnas ◽  
Gene Regulatory ◽  
Evolutionary Consequences

Abstract A striking feature of micro-RNAs is that they are often clustered in the genomes of animals. The functional and evolutionary consequences of this clustering remain obscure. Here, we investigated a micro-RNA cluster miR-6/5/4/286/3/309 that is conserved across drosophilid lineages. Small RNA sequencing revealed expression of this micro-RNA cluster in Drosophila melanogaster leg discs, and conditional overexpression of the whole cluster resulted in leg appendage shortening. Transgenic overexpression lines expressing different combinations of micro-RNA cluster members were also constructed. Expression of individual micro-RNAs from the cluster resulted in a normal wild-type phenotype, but either the expression of several ancient micro-RNAs together (miR-5/4/286/3/309) or more recently evolved clustered micro-RNAs (miR-6-1/2/3) can recapitulate the phenotypes generated by the whole-cluster overexpression. Screening of transgenic fly lines revealed downregulation of leg-patterning gene cassettes in generation of the leg-shortening phenotype. Furthermore, cell transfection with different combinations of micro-RNA cluster members revealed a suite of downstream genes targeted by all cluster members, as well as complements of targets that are unique for distinct micro-RNAs. Considered together, the micro-RNA targets and the evolutionary ages of each micro-RNA in the cluster demonstrate the importance of micro-RNA clustering, where new members can reinforce and modify the selection forces on both the cluster regulation and the gene regulatory network of existing micro-RNAs. Key words: micro-RNA, cluster, evolution.

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