We have observed that the basidiomycete Ustilago maydis can be partially or completely resistant to antibiotics when grown in defined growth media. In synthetic medium based on the fully defined mixture of simple organic compounds and salts U. maydis displays near wild-type growth at concentrations of hygromycin that effectively kill cells in complex nutrient media. The antibiotics geneticin, nourseothricin and phleomycin had similar effects. In contrast, the fungicide carboxin was equally effective in all growth media tested. Our observations could guide selection of growth media for genetic transformation of Ustilago and other fungi when sensitivity to common antibiotics is used as a selectable marker. Creative Commons License This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. This regular paper is available in Fungal Genetics Reports: http://newprairiepress.org/fgr/vol62/iss1/3 8 Differential sensitivity of Ustilago maydis to fungal antibiotics on simple and complex media Anju Verma, Tamas Kapros, and Jakob H. Waterborg * 1 Division of Plant Sciences and Bond Life Sciences Center, University of Missouri-Columbia, Columbia, MO 65211, USA; 2 Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri-Kansas City, Kansas City, MO 64110, USA. * Corresponding author Fungal Genetics Reports 62:813 We have observed that the basidiomycete Ustilago maydis can be partially or completely resistant to antibiotics when grown in defined growth media. In synthetic medium based on the fully defined mixture of simple organic compounds and salts U. maydis displays near wild-type growth at concentrations of hygromycin that effectively kill cells in complex nutrient media. The antibiotics geneticin, nourseothricin and phleomycin had similar effects. In contrast, the fungicide carboxin was equally effective in all growth media tested. Our observations could guide selection of growth media for genetic transformation of Ustilago and other fungi when sensitivity to common antibiotics is used as a selectable marker. Introduction Like many other laboratories we have successfully used the fungicide carboxin as a selectable marker in gene transformation (Brachmann et al., 2004; Brachmann et al., 2001; Fernandez-Alvarez et al., 2009; Kojic and Holloman, 2000; Topp et al., 2002) to knock-out variant-specifically histone H3 isotype loci (Verma et al., 2011). After transformation, transformants were cultured under continued selection until stable, non-heterokaryon clones could be isolated (Verma et al., 2011). These cultures were maintained on synthetic minimal media consisting of yeast nitrogen base (YNB), a standard mixture of small organic compounds and salts, with glucose (SD). When attempting to use other standard antibiotic selectable markers such as hygromycin, geneticin, nourseothricin and phleomycin (Brachmann et al., 2004; Kamper, 2004; Kojic and Holloman, 2000), selection was not observed. Other l
我们观察到担子菌麦氏黑穗病菌在确定的培养基中生长时可以部分或完全耐抗生素。在基于简单有机化合物和盐的完全确定的混合物的合成培养基中，在有效杀死复杂营养培养基中细胞的水霉素浓度下，U. maydis显示出接近野生型的生长。抗生素遗传素、诺斯霍奇霉素和静脉霉素具有相似的效果。相比之下，杀菌剂carboxin在所有培养基中都同样有效。我们的观察结果可以指导黑穗病菌和其他真菌遗传转化培养基的选择，当对常见抗生素的敏感性作为选择标记时。本作品采用知识共享署名-相同方式共享4.0许可协议。本文发表于《真菌遗传学报告》:http://newprairiepress.org/fgr/vol62/iss1/3 8简易和复杂培养基上黑孢黑菌对真菌抗生素的敏感性差异Anju Verma, Tamas Kapros，和Jakob H. Waterborg * 1密苏里大学哥伦比亚分校植物科学和Bond生命科学中心，哥伦比亚，MO 65211，美国;2密苏里大学堪萨斯城分校生物科学学院细胞生物学与生物物理学部，密苏里州堪萨斯城64110*通讯作者真菌遗传学报告62:813我们观察到担子菌黑穗病菌(Ustilago maydis)在特定的培养基中生长时可以部分或完全耐抗生素。在基于简单有机化合物和盐的完全确定的混合物的合成培养基中，在有效杀死复杂营养培养基中细胞的水霉素浓度下，U. maydis显示出接近野生型的生长。抗生素遗传素、诺斯霍奇霉素和静脉霉素具有相似的效果。相比之下，杀菌剂carboxin在所有培养基中都同样有效。我们的观察结果可以指导黑穗病菌和其他真菌遗传转化培养基的选择，当对常见抗生素的敏感性作为选择标记时。像许多其他实验室一样，我们已经成功地使用杀菌剂carboxin作为基因转化的选择标记(Brachmann et al.， 2004;Brachmann et al.， 2001;Fernandez-Alvarez et al.， 2009;Kojic and Holloman, 2000;Topp et al.， 2002)敲除变异特异性组蛋白H3同型位点(Verma et al.， 2011)。转化后，继续选择培养转化子，直到可以分离出稳定的非异核克隆(Verma et al.， 2011)。这些培养维持在由酵母氮碱(YNB)组成的合成最小培养基上，这是一种小有机化合物和盐的标准混合物，与葡萄糖(SD)。当尝试使用其他标准的抗生素选择性标记物时，如潮霉素、遗传素、诺斯红霉素和静脉霉素(Brachmann et al.， 2004;坎普,2004;Kojic和Holloman, 2000)，没有观察到选择。其他实验室也广泛使用这些标记来产生黑穗病菌的遗传转化体，但他们使用的是化学复杂的培养基，主要是酵母提取物和杆菌肽的混合物，并补充葡萄糖(YPD)或蔗糖(YPS) (Berndt等人，2010;Brachmann et al.， 2004;Brachmann et al.， 2001;Lovely et al.， 2011;Tsukuda et al.， 1988)，还有Complete Medium (Garcia-Pedrajas et al.， 2008;霍利迪,1974;Lee et al.， 1999)或马铃薯葡萄糖(Brachmann et al.， 2004;Heidenreich et al.， 2008;Zameitat et al.， 2007)。在我们的手中，这些复杂的介质，特别是YPS，可以有效地用于抗生素的选择。然而，我们在这里报道，在简单的SD培养基中，氨基糖苷类和糖肽类抗生素，都比碳毒素更大，更亲水，大部分或完全无效。我们推测这些抗生素的摄取依赖于简单SD培养基中不存在的黑穗病菌细胞膜转运机制。如果想要在基于ynb的合成最小培养基中创建营养缺陷突变体，例如必需氨基酸如亮氨酸(Fotheringham和Holloman, 1990)或核苷酸碱基如腺嘌呤(Verma et al.， 2016)，则该观察结果会构成障碍。材料和方法简单的合成培养基SD和SS分别由6.7 g不含氨基酸的酵母氮碱(YNB) (Fisher Scientific, BD Difco)和20 g葡萄糖(glucose)或蔗糖(Sigma-Aldrich)在1 L水中组成。加入50 mM琥珀酸制备pH缓冲SD培养基(SDS)，并于2017年由新草原出版社出版。半确定的复合生长培养基YPD和YPS2分别含有10 g酵母提取物(BD Difco)、20 g杆菌肽(BD Difco)和20 g葡萄糖或蔗糖，在1 L水中。除了这种丰富的复杂培养基(Tsukuda et al.， 1988)，我们还测试了较差的配方YPS1，每L只有4克酵母提取物和4克杆菌肽(Brachmann et al.， 2004)。 例如，在黑穗病菌的原生质体转化过程中，高浓度山梨醇是原生质体稳定所必需的，但它对黑穗病菌原生质体的稳定性有很大的影响
{"title":"Differential sensitivity of Ustilago maydis to fungal antibiotics on simple and complex media","authors":"A. Verma, T. Kapros, J. H. Waterborg","doi":"10.4148/1941-4765.1002","DOIUrl":"https://doi.org/10.4148/1941-4765.1002","url":null,"abstract":"We have observed that the basidiomycete Ustilago maydis can be partially or completely resistant to antibiotics when grown in defined growth media. In synthetic medium based on the fully defined mixture of simple organic compounds and salts U. maydis displays near wild-type growth at concentrations of hygromycin that effectively kill cells in complex nutrient media. The antibiotics geneticin, nourseothricin and phleomycin had similar effects. In contrast, the fungicide carboxin was equally effective in all growth media tested. Our observations could guide selection of growth media for genetic transformation of Ustilago and other fungi when sensitivity to common antibiotics is used as a selectable marker. Creative Commons License This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. This regular paper is available in Fungal Genetics Reports: http://newprairiepress.org/fgr/vol62/iss1/3 8 Differential sensitivity of Ustilago maydis to fungal antibiotics on simple and complex media Anju Verma, Tamas Kapros, and Jakob H. Waterborg * 1 Division of Plant Sciences and Bond Life Sciences Center, University of Missouri-Columbia, Columbia, MO 65211, USA; 2 Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri-Kansas City, Kansas City, MO 64110, USA. * Corresponding author Fungal Genetics Reports 62:813 We have observed that the basidiomycete Ustilago maydis can be partially or completely resistant to antibiotics when grown in defined growth media. In synthetic medium based on the fully defined mixture of simple organic compounds and salts U. maydis displays near wild-type growth at concentrations of hygromycin that effectively kill cells in complex nutrient media. The antibiotics geneticin, nourseothricin and phleomycin had similar effects. In contrast, the fungicide carboxin was equally effective in all growth media tested. Our observations could guide selection of growth media for genetic transformation of Ustilago and other fungi when sensitivity to common antibiotics is used as a selectable marker. Introduction Like many other laboratories we have successfully used the fungicide carboxin as a selectable marker in gene transformation (Brachmann et al., 2004; Brachmann et al., 2001; Fernandez-Alvarez et al., 2009; Kojic and Holloman, 2000; Topp et al., 2002) to knock-out variant-specifically histone H3 isotype loci (Verma et al., 2011). After transformation, transformants were cultured under continued selection until stable, non-heterokaryon clones could be isolated (Verma et al., 2011). These cultures were maintained on synthetic minimal media consisting of yeast nitrogen base (YNB), a standard mixture of small organic compounds and salts, with glucose (SD). When attempting to use other standard antibiotic selectable markers such as hygromycin, geneticin, nourseothricin and phleomycin (Brachmann et al., 2004; Kamper, 2004; Kojic and Holloman, 2000), selection was not observed. Other l","PeriodicalId":12490,"journal":{"name":"Fungal Genetics Reports","volume":"1 1","pages":"8-13"},"PeriodicalIF":0.0,"publicationDate":"2016-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"83009112","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Zachary J Smith, Stacy Bedore, Stephanie Spingler, Thomas M Hammond
This report describes the construction and characterization of mus-51RIP70 , an allele for high-efficiency targeted integration of transgenes into the genome of the model eukaryote Neurospora crassa. Two of the mus-51RIP70 strains investigated in this work (RZS27.10 and RZS27.18) can be obtained from the Fungal Genetics Stock Center. The two deposited strains are, to our knowledge, genetically identical and neither one is preferred over the other for use in Neurospora research.
{"title":"A <i>mus-51</i> RIP allele for transformation of <i>Neurospora crassa</i>.","authors":"Zachary J Smith, Stacy Bedore, Stephanie Spingler, Thomas M Hammond","doi":"10.4148/1941-4765.1001","DOIUrl":"https://doi.org/10.4148/1941-4765.1001","url":null,"abstract":"<p><p>This report describes the construction and characterization of <i>mus-51<sup>RIP70</sup></i> , an allele for high-efficiency targeted integration of transgenes into the genome of the model eukaryote <i>Neurospora crassa</i>. Two of the <i>mus-51<sup>RIP70</sup></i> strains investigated in this work (RZS27.10 and RZS27.18) can be obtained from the Fungal Genetics Stock Center. The two deposited strains are, to our knowledge, genetically identical and neither one is preferred over the other for use in <i>Neurospora</i> research.</p>","PeriodicalId":12490,"journal":{"name":"Fungal Genetics Reports","volume":"62 ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2016-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5515490/pdf/nihms879885.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35188965","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Neurospora has been studied extensively to understand various phenomenon of fungal growth and morphogenesis. Fungal morphogenesis is a complex process which includes important aspects like polarized extension of the hyphal tip and hyphal branching. Observations at the tip have shown that growth vesicles arrive at the tip from proximal locations, and then seem to be distributed into the tip dome by the spitzenkorper. No specific mechanism has been defined for branching. Several theories have been proposed for branching that can be broadly divided into two groups. One group supports the mechanism involving origin of branch initiation factors at the hyphal tips (Bachewich and Heath, 1997; Kaminskyj and Heath, 1996; Riquelme and Bartnicki-Garcia, 2004; Watters and Griffiths, 2001). Other group suggests that the signals for branch initiation originate from within the colony or mycelial body (Prosser and Trinci, 1979; Trinci, 1974; Watters, 2006). Watters et al., (2000) proposed that the branching initiation is not completely controlled by the tip but, to some extent by factors occurring at previous branch point. Trinci (1974) proposed that mutation or factor which reduces the maximum rate of tip extension without disturbing the rate of vesicle production would results in an increase in the frequency of branch initiation without reducing the overall rate of hypha formation. Recently it has been found that the fungal growth rate show relationship with the frequency of hyphal branching (Watters et al., 2008, 2011). In this paper we describe a naturally occurring mutant of N. intermedia which shows defect in branching and the inheritance of this defect in the progeny.
人们对神经孢子菌进行了广泛的研究，以了解真菌生长和形态发生的各种现象。真菌形态发生是一个复杂的过程，包括菌丝尖端的极化延伸和菌丝分支等重要方面。在尖端的观察表明，生长囊泡从近端到达尖端，然后似乎通过spitzenkorper分布到尖端穹窿中。没有为分支定义特定的机制。关于分支，人们提出了几种理论，大致可以分为两类。一个小组支持涉及菌丝尖端分支起始因子起源的机制(Bachewich和Heath, 1997;Kaminskyj and Heath, 1996;Riquelme and Bartnicki-Garcia, 2004;Watters and Griffiths, 2001)。另一组认为分支形成的信号来自菌落或菌丝体内部(Prosser和Trinci, 1979;Trinci, 1974;继续萎缩,2006)。Watters et al.，(2000)提出分支的发生并不完全由尖端控制，而是在一定程度上由前一个分支点发生的因素控制。Trinci(1974)提出，在不干扰囊泡产生速率的情况下，降低最大尖端延伸速率的突变或因子会导致分支起始频率的增加，而不会降低菌丝形成的总体速率。最近发现真菌的生长速度与菌丝分支的频率有关(Watters et al.， 2008, 2011)。本文描述了一种具有分枝缺陷的自然突变体，以及这种缺陷在后代中的遗传。
{"title":"Identification and characterization of a new branching mutant of Neurospora intermedia from nature","authors":"A. Mukati, H. Vyas, A. Vyas","doi":"10.4148/1941-4765.1004","DOIUrl":"https://doi.org/10.4148/1941-4765.1004","url":null,"abstract":"Neurospora has been studied extensively to understand various phenomenon of fungal growth and morphogenesis. Fungal morphogenesis is a complex process which includes important aspects like polarized extension of the hyphal tip and hyphal branching. Observations at the tip have shown that growth vesicles arrive at the tip from proximal locations, and then seem to be distributed into the tip dome by the spitzenkorper. No specific mechanism has been defined for branching. Several theories have been proposed for branching that can be broadly divided into two groups. One group supports the mechanism involving origin of branch initiation factors at the hyphal tips (Bachewich and Heath, 1997; Kaminskyj and Heath, 1996; Riquelme and Bartnicki-Garcia, 2004; Watters and Griffiths, 2001). Other group suggests that the signals for branch initiation originate from within the colony or mycelial body (Prosser and Trinci, 1979; Trinci, 1974; Watters, 2006). Watters et al., (2000) proposed that the branching initiation is not completely controlled by the tip but, to some extent by factors occurring at previous branch point. Trinci (1974) proposed that mutation or factor which reduces the maximum rate of tip extension without disturbing the rate of vesicle production would results in an increase in the frequency of branch initiation without reducing the overall rate of hypha formation. Recently it has been found that the fungal growth rate show relationship with the frequency of hyphal branching (Watters et al., 2008, 2011). In this paper we describe a naturally occurring mutant of N. intermedia which shows defect in branching and the inheritance of this defect in the progeny.","PeriodicalId":12490,"journal":{"name":"Fungal Genetics Reports","volume":"1 1","pages":"1-8"},"PeriodicalIF":0.0,"publicationDate":"2015-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"77196450","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Joshua C. Springer, A. Baines, Matthew T. Chansler, A. Jarosz
Isolates of the Chestnut blight pathogen, Cryphonectria parasitica, from six populations in Michigan, were stored in the late 1990s as agar plugs of mycelium in vials of sterile water held at room temperature. Approximately 29% of the fungal isolates were infected with mycoviruses at the time of storage. Each isolate was tested for revivification effectiveness by taking aliquots from vials filled with agar plugs of C. parasitica and sterile water and plating onto potato dextrose agar. Average revivification success was 70.5% across populations with a range of 33—84% within populations. In situations where vials had dried out during storage, success was low (4%), while success for vials that retained sterile water averaged 90%. Most importantly however, is the loss of mycoviruses from stored isolates; only 2 of 119 stored mycovirus infected isolates still contained mycoviruses after storage, suggesting that the double-stranded RNA mycoviruses are degraded during storage. Creative Commons License This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. This regular paper is available in Fungal Genetics Reports: http://newprairiepress.org/fgr/vol60/iss1/2
{"title":"Evaluating the long-term storage of Cryphonectria parasitica","authors":"Joshua C. Springer, A. Baines, Matthew T. Chansler, A. Jarosz","doi":"10.4148/1941-4765.1007","DOIUrl":"https://doi.org/10.4148/1941-4765.1007","url":null,"abstract":"Isolates of the Chestnut blight pathogen, Cryphonectria parasitica, from six populations in Michigan, were stored in the late 1990s as agar plugs of mycelium in vials of sterile water held at room temperature. Approximately 29% of the fungal isolates were infected with mycoviruses at the time of storage. Each isolate was tested for revivification effectiveness by taking aliquots from vials filled with agar plugs of C. parasitica and sterile water and plating onto potato dextrose agar. Average revivification success was 70.5% across populations with a range of 33—84% within populations. In situations where vials had dried out during storage, success was low (4%), while success for vials that retained sterile water averaged 90%. Most importantly however, is the loss of mycoviruses from stored isolates; only 2 of 119 stored mycovirus infected isolates still contained mycoviruses after storage, suggesting that the double-stranded RNA mycoviruses are degraded during storage. Creative Commons License This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. This regular paper is available in Fungal Genetics Reports: http://newprairiepress.org/fgr/vol60/iss1/2","PeriodicalId":12490,"journal":{"name":"Fungal Genetics Reports","volume":"12 1","pages":"11-15"},"PeriodicalIF":0.0,"publicationDate":"2013-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"89916058","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Nicholas Harrison, Brad Cavinder, J. Townsend, F. Trail
Methods to streamline functional studies of large numbers of genes are essential to fully utilize the significant genomic resources now available for fungi. Fusion PCR is often used to join pieces of DNA together, particularly in the construction of DNA fragments for gene replacement in fungi. Here we present high-efficiency primers which reliably direct fusion and amplification to generate constructs for gene knockouts.
{"title":"Optimized primers and other critical conditions for efficient fusion PCR to generate knockout vectors in filamentous fungi.","authors":"Nicholas Harrison, Brad Cavinder, J. Townsend, F. Trail","doi":"10.4148/1941-4765.1006","DOIUrl":"https://doi.org/10.4148/1941-4765.1006","url":null,"abstract":"Methods to streamline functional studies of large numbers of genes are essential to fully utilize the significant genomic resources now available for fungi. Fusion PCR is often used to join pieces of DNA together, particularly in the construction of DNA fragments for gene replacement in fungi. Here we present high-efficiency primers which reliably direct fusion and amplification to generate constructs for gene knockouts.","PeriodicalId":12490,"journal":{"name":"Fungal Genetics Reports","volume":"33 1","pages":"1-10"},"PeriodicalIF":0.0,"publicationDate":"2013-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"76233216","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Genomic DNA sequence was determined for the putative Neurospora crassa ABC transporter NCU09975 from several different classical mutant strains including several acriflavin resistant mutants. The sensitivity of these strains to acriflavin was tested. While the open reading frame NCU09975 has multiple polymorphisms in strains sequenced for other purposes, none of the acriflavin resistant classical mutants tested had polymorphisms in the NCU09975 coding region or in the 195 bases upstream of the translation start site. Creative Commons License This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. This regular paper is available in Fungal Genetics Reports: http://newprairiepress.org/fgr/vol59/iss1/4 Fungal Genetics Reports 59, 2012. 26 Analysis of the DNA sequence of the putative ABC transporter NCU09975 in Neurospora crassa strains carrying acriflavin resistance markers. Aric E. Wiest, Sarah Koch and Kevin McCluskey. Fungal Genetics Stock Center, University of MissouriKansas City, Kansas City, MO 94110 USA Fungal Genet Reports 59: 26 29 Genomic DNA sequence was determined for the putative Neurospora crassa ABC transporter NCU09975 from several different classical mutant strains including several acriflavin resistant mutants. The sensitivity of these strains to acriflavin was tested. While the open reading frame NCU09975 has multiple polymorphisms in strains sequenced for other purposes, none of the acriflavin resistant classical mutants tested had polymorphisms in the NCU09975 coding region or in the 195 bases upstream of the translation start site. Introduction While years of mutagenesis and subsequent characterization in Neurospora crassa produced one of the most densely saturated genetic maps (Perkins et al. 2001), many of the classical mutants are not yet associated with open reading frames from the genome sequence (Galagan et al. 2003). Among these are acriflavin resistant mutants which have been assigned to seven different loci. A subset of these genes also confer resistance to multiple related drugs, including acridine orange, malachite green, and aminotriazole (Akiyama and Nakashima 1996). Moreover some acriflavin resistance genes are dominant while others are recessive. Because we wanted to find a dominant selectable marker for transformation of Neurospora and because the abc-3-like ORF NCU09975 had a large number of polymorphisms in its primary sequence among a group strains subject to whole genome sequence (McCluskey et al. 2011) we undertook to characterize this locus in strains carrying the acriflavin resistance markers acr-1 and Acr-3. We additionally validated the acriflavin sensitivity in the classical mutant strains carrying polymorphisms in NCU09975. Materials and methods Strains were cultured on Vogel's minimal medium (Vogel 1956) using standard practices (Davis and De Serres 1970) (Table 1). Table 1. Strains and their characteristics FGSC number Genotype Marker Location Acriflavin sensitivity 2489 m
测定了几种不同经典突变菌株(包括几种抗吖啶黄素突变株)中假定的粗神经孢子虫ABC转运体NCU09975的基因组DNA序列。测定了这些菌株对吖啶黄素的敏感性。虽然开放阅读框NCU09975在用于其他目的测序的菌株中具有多个多态性，但测试的抗吖啶黄素经典突变体在NCU09975编码区或翻译起始位点上游195个碱基中均没有多态性。本作品采用知识共享署名-相同方式共享4.0许可协议。这篇常规论文可在真菌遗传学报告:http://newprairiepress.org/fgr/vol59/iss1/4真菌遗传学报告59,2012。26携带吖啶黄素抗性标记的粗神经孢子虫ABC转运体NCU09975的DNA序列分析。Aric E. Wiest, Sarah Koch和Kevin McCluskey。真菌基因报告59:26 29从几种不同的经典突变菌株(包括几种抗吖啶黄素突变菌株)中确定了假定的粗神经孢子虫ABC转运体NCU09975的基因组DNA序列。测定了这些菌株对吖啶黄素的敏感性。虽然开放阅读框NCU09975在用于其他目的测序的菌株中具有多个多态性，但测试的抗吖啶黄素经典突变体在NCU09975编码区或翻译起始位点上游195个碱基中均没有多态性。虽然对粗神经孢子虫多年的诱变和随后的表征产生了最密集饱和的遗传图谱之一(Perkins et al. 2001)，但许多经典突变体尚未与基因组序列的开放阅读框相关联(Galagan et al. 2003)。其中有抗吖啶黄素的突变体，它们分布在7个不同的基因座上。这些基因的一个子集还赋予对多种相关药物的抗性，包括吖啶橙、孔雀石绿和氨基三唑(Akiyama和Nakashima 1996)。此外，一些抗吖啶黄素基因是显性的，而另一些是隐性的。由于我们希望找到神经孢子虫转化的显性选择标记，并且由于abc-3样ORF NCU09975在全基因组测序的一组菌株中其一级序列存在大量多态性(McCluskey et al. 2011)，我们在携带吖啶黄素抗性标记acr-1和Acr-3的菌株中对该位点进行了表征。我们还验证了NCU09975携带多态性的经典突变菌株对吖啶黄素的敏感性。材料和方法采用标准方法(Davis and De Serres 1970)在Vogel's minimal培养基(Vogel 1956)上培养菌株(表1)。菌株及其特征FGSC数量基因型标记位置黄酮类敏感性2489席-a IL敏感1215 Acr-3席-a IL耐IL 1209 Acr-3席-a IL耐IL 875 acr-1席-a IL敏感305 amyc席-a IL IL敏感1363 smco-1席-a IL敏感3921 tng席-a IL;敏感7022字段IVR;《真菌遗传学报告》，2012年第59期，新草原出版社出版。27 .加入无菌培养基前，将吖啶黄素溶于水中，过滤灭菌。它被储存在黑暗中，并定期准备新鲜的库存。将10毫升新鲜制备的分生孢子和菌丝片段悬浮液(约103 cfu/ ml)移液到10 × 75 mm玻璃培养管中的琼脂固化培养基表面，进行吖啶黄素敏感性测试。分别在2天、4天和10天后记录结果。使用ZR真菌DNA试剂盒(zimo Research, Irvine, CA)从23天龄的液体摇培养物中提取基因组DNA。使用该ORF特异性引物扩增NCU09975 DNA如图1所示(表2)。引物1F和5R一起扩增2848个碱基片段，包括起始密码子和195个上游碱基。引物7F和11R用于扩增2878个碱基片段，包括终止密码子和下游667个碱基(图1)。这些重叠370个碱基的大片段在UMKC生物科学学院的核心设施上使用Applied Biosystems 3100遗传分析仪(Foster City, USA)直接测序。测序和分析使用Sequencher (Gene Codes Corporation, Ann Arbor, USA)。
{"title":"Analysis of the DNA sequence of the putative ABC transporter NCU09975 in Neurospora crassa strains carrying acriflavin resistance markers.","authors":"A. Wiest, S. Koch, K. McCluskey","doi":"10.4148/1941-4765.1012","DOIUrl":"https://doi.org/10.4148/1941-4765.1012","url":null,"abstract":"Genomic DNA sequence was determined for the putative Neurospora crassa ABC transporter NCU09975 from several different classical mutant strains including several acriflavin resistant mutants. The sensitivity of these strains to acriflavin was tested. While the open reading frame NCU09975 has multiple polymorphisms in strains sequenced for other purposes, none of the acriflavin resistant classical mutants tested had polymorphisms in the NCU09975 coding region or in the 195 bases upstream of the translation start site. Creative Commons License This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. This regular paper is available in Fungal Genetics Reports: http://newprairiepress.org/fgr/vol59/iss1/4 Fungal Genetics Reports 59, 2012. 26 Analysis of the DNA sequence of the putative ABC transporter NCU09975 in Neurospora crassa strains carrying acriflavin resistance markers. Aric E. Wiest, Sarah Koch and Kevin McCluskey. Fungal Genetics Stock Center, University of MissouriKansas City, Kansas City, MO 94110 USA Fungal Genet Reports 59: 26 29 Genomic DNA sequence was determined for the putative Neurospora crassa ABC transporter NCU09975 from several different classical mutant strains including several acriflavin resistant mutants. The sensitivity of these strains to acriflavin was tested. While the open reading frame NCU09975 has multiple polymorphisms in strains sequenced for other purposes, none of the acriflavin resistant classical mutants tested had polymorphisms in the NCU09975 coding region or in the 195 bases upstream of the translation start site. Introduction While years of mutagenesis and subsequent characterization in Neurospora crassa produced one of the most densely saturated genetic maps (Perkins et al. 2001), many of the classical mutants are not yet associated with open reading frames from the genome sequence (Galagan et al. 2003). Among these are acriflavin resistant mutants which have been assigned to seven different loci. A subset of these genes also confer resistance to multiple related drugs, including acridine orange, malachite green, and aminotriazole (Akiyama and Nakashima 1996). Moreover some acriflavin resistance genes are dominant while others are recessive. Because we wanted to find a dominant selectable marker for transformation of Neurospora and because the abc-3-like ORF NCU09975 had a large number of polymorphisms in its primary sequence among a group strains subject to whole genome sequence (McCluskey et al. 2011) we undertook to characterize this locus in strains carrying the acriflavin resistance markers acr-1 and Acr-3. We additionally validated the acriflavin sensitivity in the classical mutant strains carrying polymorphisms in NCU09975. Materials and methods Strains were cultured on Vogel's minimal medium (Vogel 1956) using standard practices (Davis and De Serres 1970) (Table 1). Table 1. Strains and their characteristics FGSC number Genotype Marker Location Acriflavin sensitivity 2489 m","PeriodicalId":12490,"journal":{"name":"Fungal Genetics Reports","volume":"164 1","pages":"26-29"},"PeriodicalIF":0.0,"publicationDate":"2012-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"73376144","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
M. Déquard-Chablat, Tan-Trung Nguyen, V. Contamine, S. H. Denmat, F. Malagnac
Because, in filamentous fungi, integration of transgenes proceeds mainly through nonhomologous recombination, transformation results in a random distribution of these DNA sequences. Moreover, they are often found as multiple copies clustered at a single locus. Therefore, for a given transgene, expression may be highly variable among independent transformants. To get rid of both multiple copies and position effects, due to the impact of the chromatin structure upon transgene expression, we constructed tools to specifically target single copies at two well-defined loci, Pa_2_3690 and Pa_4_5450 (Espagne et al., 2008). These genes have been chosen for the following reasons: (1) they have a relatively stable expression during the entire Podospora’s life cycle, (2) complete deletion of their ORF does not lead to an abnormal phenotype (Bidard et al., 2011), (3) they are located on two different chromosomes and (4) their second division segregation (SDS) frequency is 80% and 90%, respectively. Besides, a highly efficient homologous recombination ΔPaKu70 strain is available (El-Khoury et al., 2008), which makes gene replacement fairly easy. Taking advantage of this strain, we designed two plasmid tools to target genomic integration of any DNA fragment.
因为，在丝状真菌中，转基因的整合主要通过非同源重组进行，转化导致这些DNA序列的随机分布。此外，它们经常在单个位点上聚集多个拷贝。因此，对于给定的转基因，在独立的转化子之间表达可能是高度可变的。由于染色质结构对转基因表达的影响，为了消除多拷贝和位置效应，我们构建了专门针对两个定义明确的位点Pa_2_3690和Pa_4_5450的单拷贝的工具(Espagne et al.， 2008)。选择这些基因的原因如下:(1)它们在整个Podospora的生命周期中表达相对稳定;(2)它们的ORF完全缺失不会导致表型异常(Bidard et al.， 2011);(3)它们位于两条不同的染色体上;(4)它们的二次分裂分离(SDS)频率分别为80%和90%。此外，有一种高效的同源重组ΔPaKu70菌株(El-Khoury et al.， 2008)，使得基因替换相当容易。利用该菌株的优势，我们设计了两个质粒工具，用于任何DNA片段的基因组整合。
{"title":"Efficient tools to target DNA to Podospora anserina","authors":"M. Déquard-Chablat, Tan-Trung Nguyen, V. Contamine, S. H. Denmat, F. Malagnac","doi":"10.4148/1941-4765.1011","DOIUrl":"https://doi.org/10.4148/1941-4765.1011","url":null,"abstract":"Because, in filamentous fungi, integration of transgenes proceeds mainly through nonhomologous recombination, transformation results in a random distribution of these DNA sequences. Moreover, they are often found as multiple copies clustered at a single locus. Therefore, for a given transgene, expression may be highly variable among independent transformants. To get rid of both multiple copies and position effects, due to the impact of the chromatin structure upon transgene expression, we constructed tools to specifically target single copies at two well-defined loci, Pa_2_3690 and Pa_4_5450 (Espagne et al., 2008). These genes have been chosen for the following reasons: (1) they have a relatively stable expression during the entire Podospora’s life cycle, (2) complete deletion of their ORF does not lead to an abnormal phenotype (Bidard et al., 2011), (3) they are located on two different chromosomes and (4) their second division segregation (SDS) frequency is 80% and 90%, respectively. Besides, a highly efficient homologous recombination ΔPaKu70 strain is available (El-Khoury et al., 2008), which makes gene replacement fairly easy. Taking advantage of this strain, we designed two plasmid tools to target genomic integration of any DNA fragment.","PeriodicalId":12490,"journal":{"name":"Fungal Genetics Reports","volume":"305 1","pages":"21-25"},"PeriodicalIF":0.0,"publicationDate":"2012-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"79819811","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
T. Radic, S. Gastaldello, J. Diegmann, T. Roenneberg
The ascomycete Neurospora crassa is classical model organisms in biology. So far, a phylogenetic analysis based on genomic sequences of four non-functional nuclear loci has been reported for 44 natural isolates of N. crassa. Three subgroups (clades) with a distinct geographical distribution have been identified: clade A (Caribbean Basin and Ivory Coast), clade B (Europe, Ivory Coast and India), and clade C (India). Here, we report the results of a phylogenetic analysis of 16 additional isolates. Six of these were from the Caribbean Basin, eight from Europe and one from Pakistan and one from Thailand. The previously described clades and their geographical distribution were generally confirmed. All Caribbean isolates belonged to clade A and all European isolates belonged to clade B, with the exception of one isolate from Italy, which also belonged to clade A, suggesting a transport from the Caribbean Basin or the Ivory Coast to Europe. Interestingly, the isolates from Pakistan and Thailand were found in a separate group, basal to all other clades. Their phylogenetic classification is not yet clear as they might belong to N. crassa but as well to N. perkinsii, potentially representing yet undescribed phylogenetic groups or species of Neurospora, or hybrids. Authors Tanja Radic, Silvia Gastaldello, Julia Diegmann, and Till Roenneberg This regular paper is available in Fungal Genetics Reports: https://newprairiepress.org/fgr/vol59/iss1/2
粗神经孢子子囊菌是生物学中经典的模式生物。迄今为止，已报道了44个自然分离株的4个非功能性核位点的基因组序列系统发育分析。已经确定了具有不同地理分布的三个亚群(分支):分支a(加勒比盆地和象牙海岸)，分支B(欧洲，象牙海岸和印度)和分支C(印度)。在这里，我们报告了另外16个分离株的系统发育分析结果。其中6人来自加勒比海盆地，8人来自欧洲，1人来自巴基斯坦，1人来自泰国。前人所描述的进化支及其地理分布基本得到了证实。所有加勒比分离株都属于进化支A，所有欧洲分离株都属于进化支B，除了意大利的一株分离株，它也属于进化支A，这表明该病毒是从加勒比盆地或科特迪瓦传播到欧洲的。有趣的是，来自巴基斯坦和泰国的分离株被发现在一个单独的群体中，是所有其他分支的基础。它们的系统发育分类尚不清楚，因为它们可能属于N. crassa，也可能属于N. perkinsii，可能代表尚未描述的系统发育类群或神经孢子虫种，或杂种。作者Tanja Radic, Silvia gastalello, Julia Diegmann和Till Roenneberg这篇常规论文可在真菌遗传学报告中找到:https://newprairiepress.org/fgr/vol59/iss1/2
{"title":"Phylogenic analysis of additional Neurospora crassa isolates","authors":"T. Radic, S. Gastaldello, J. Diegmann, T. Roenneberg","doi":"10.4148/1941-4765.1010","DOIUrl":"https://doi.org/10.4148/1941-4765.1010","url":null,"abstract":"The ascomycete Neurospora crassa is classical model organisms in biology. So far, a phylogenetic analysis based on genomic sequences of four non-functional nuclear loci has been reported for 44 natural isolates of N. crassa. Three subgroups (clades) with a distinct geographical distribution have been identified: clade A (Caribbean Basin and Ivory Coast), clade B (Europe, Ivory Coast and India), and clade C (India). Here, we report the results of a phylogenetic analysis of 16 additional isolates. Six of these were from the Caribbean Basin, eight from Europe and one from Pakistan and one from Thailand. The previously described clades and their geographical distribution were generally confirmed. All Caribbean isolates belonged to clade A and all European isolates belonged to clade B, with the exception of one isolate from Italy, which also belonged to clade A, suggesting a transport from the Caribbean Basin or the Ivory Coast to Europe. Interestingly, the isolates from Pakistan and Thailand were found in a separate group, basal to all other clades. Their phylogenetic classification is not yet clear as they might belong to N. crassa but as well to N. perkinsii, potentially representing yet undescribed phylogenetic groups or species of Neurospora, or hybrids. Authors Tanja Radic, Silvia Gastaldello, Julia Diegmann, and Till Roenneberg This regular paper is available in Fungal Genetics Reports: https://newprairiepress.org/fgr/vol59/iss1/2","PeriodicalId":12490,"journal":{"name":"Fungal Genetics Reports","volume":"68 1","pages":"13-20"},"PeriodicalIF":0.0,"publicationDate":"2012-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"75393348","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Kylie J. Boyce, Hayley E. Bugeja, Harshini Weerasinghe, Michael Payne, Lena Schreider
P. marneffei has been established as an experimentally amenable system to study morphogenesis and pathogenicity. This paper describes the development of a number of tools, including numerous selectable markers, to expand the ease with which it can be genetically manipulated. Combined with strains engineered for homologous recombination of exogenous DNA, these tools facilitate efficient molecular genetic studies.
{"title":"Strategies for the molecular genetic manipulation and visualization of the human fungal pathogen Penicillium marneffei","authors":"Kylie J. Boyce, Hayley E. Bugeja, Harshini Weerasinghe, Michael Payne, Lena Schreider","doi":"10.4148/1941-4765.1009","DOIUrl":"https://doi.org/10.4148/1941-4765.1009","url":null,"abstract":"P. marneffei has been established as an experimentally amenable system to study morphogenesis and pathogenicity. This paper describes the development of a number of tools, including numerous selectable markers, to expand the ease with which it can be genetically manipulated. Combined with strains engineered for homologous recombination of exogenous DNA, these tools facilitate efficient molecular genetic studies.","PeriodicalId":12490,"journal":{"name":"Fungal Genetics Reports","volume":"5 1","pages":"1-12"},"PeriodicalIF":0.0,"publicationDate":"2012-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"81874209","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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