Here, we report the construction of two transformation vectors, pD-NAT1 and pG-NAT1, carrying the nat1 gene encoding the nourseothricin acetyltransferase. The nat1 gene is expressed under the control of the Aspergillus nidulans trpC promoter and thus can be used as a dominant drug-resistance marker for the DNA-mediated transformation of filamentous fungi. The successful application of both vectors was demonstrated by transforming the homothallic ascomycete Sordaria macrospora as well as the β-lactam producer Acremonium chrysogenum. For both fungi and for both vectors, transformation frequencies were between 10 and 40 transformants per 10 µg of plasmid DNA.
{"title":"Application of the nourseothricin acetyltransferase gene (nat1) as dominant marker for the transformation of filamentous fungi","authors":"U. Kück, Birgit Hoff, Lehrstuhl für Allgemeine","doi":"10.4148/1941-4765.1106","DOIUrl":"https://doi.org/10.4148/1941-4765.1106","url":null,"abstract":"Here, we report the construction of two transformation vectors, pD-NAT1 and pG-NAT1, carrying the nat1 gene encoding the nourseothricin acetyltransferase. The nat1 gene is expressed under the control of the Aspergillus nidulans trpC promoter and thus can be used as a dominant drug-resistance marker for the DNA-mediated transformation of filamentous fungi. The successful application of both vectors was demonstrated by transforming the homothallic ascomycete Sordaria macrospora as well as the β-lactam producer Acremonium chrysogenum. For both fungi and for both vectors, transformation frequencies were between 10 and 40 transformants per 10 µg of plasmid DNA.","PeriodicalId":12490,"journal":{"name":"Fungal Genetics Reports","volume":"10 1","pages":"9-11"},"PeriodicalIF":0.0,"publicationDate":"2006-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"74815144","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}
The ribosomal proteins of the E. coli S4, S5 and S12 families that are part of the ribosome accuracy center control translation accuracy both in prokaryotes and eukaryotes. In Podospora anserina, genes coding for S4 and S5 have already been identified. Here, we identify the gene coding for the S12 protein homologue and show that it is identical to the genetically known AS6 gene. 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/vol53/iss1/8 26 Fungal Genetics Newsletter Podospora anserina AS6 gene encodes the cytosolic ribosomal protein of the E. coli S12 family Michelle Dequard-Chablat and Philippe Silar 1 1, 2 Institut de Génétique et Microbiologie, Bât. 400 Université de Paris 11, 91405 Orsay cedex, France. 1 UFR de Biochimie, Université de Paris 7 Denis Diderot, case 7006, 2 place Jussieu, 75005, Paris, France 2 To whom correspondence may be addressed: michelle.chablat@igmors.u-psud.fr or philippe.silar@igmors.u-psud.fr Fungal Genetics Newsletter 53: 26-29 The ribosomal proteins of the E. coli S4, S5 and S12 families that are part of the ribosome accuracy center control translation accuracy both in prokaryotes and eukaryotes. In Podospora anserina, genes coding for S4 and S5 have already been identified. Here, we identify the gene coding for the S12 protein homologue and show that it is identical to the genetically known AS6 gene. Podospora anserina has been used in intensive search of translation accuracy mutants (Coppin-Raynal et al. 1988). Several factors involved in the maintenance of accuracy have been identified in this organism including the tRNA suppressors su4-1 and su8-1 (Debuchy et al. 1985), as well as elongation factor eEF1A coded by AS4 (Silar et al. 1994), termination factors eRF1 and eRF3 coded by su1 and su2/AS2 respectively (Gagny et al. 1998), ribosomal proteins S12 coded by AS1 (Dequard-Chablat et al. 1994), S7 coded by su12 (Silar et al. 1997) and S1 coded by su3 (Silar et al. 2003). S12, S7 and S1 refer to the P. anserina numbering for ribosomal proteins (Dequard-Chablat et al. 1986) since the su12 and su3 genes code for the ribosomal proteins homologues of the E. coli S4 and S5, respectively. These two proteins are part of an accuracy center that has been conserved for more than two billion years in both prokaryotes and eukaryotes (Alksne et al. 1993). The center contains a third protein corresponding to the E. coli S12 protein, which remains to be identified in P. anserina. This protein is highly conserved and essential in all prokaryotes and eukaryotes investigated to date (Alksne et al. 1993). To identify the gene coding this protein in P. anserina, we took advantage of the availability of the complete genomic sequence of this fungus (available at http://podospora.igmors.u-psud.fr). We first located on the sequence map the gene encoding the P. anserina protein of the S1
大肠杆菌S4、S5和S12家族的核糖体蛋白作为核糖体精度中心的一部分，控制着原核生物和真核生物的翻译精度。在猪足孢子虫中，已经鉴定出编码S4和S5的基因。在这里，我们鉴定了编码S12蛋白同源物的基因，并表明它与遗传上已知的AS6基因相同。本作品采用知识共享署名-相同方式共享4.0许可协议。这篇常规论文发表在真菌遗传学报告:http://newprairiepress.org/fgr/vol53/iss1/8 26真菌遗传学通讯Podospora anserina AS6基因编码大肠杆菌S12家族的细胞质核糖体蛋白。1 UFR de Biochimie, universit<s:1> de Paris 7 Denis Diderot, case 7006, 2 place Jussieu, 75005, Paris, France 2通讯地址:michelle.chablat@igmors.u-psud.fr或philippe.silar@igmors.u-psud.fr真菌遗传学通讯53:26-29大肠杆菌S4, S5和S12家族的核糖体蛋白是核糖体准确性中心的一部分，控制着原核生物和真核生物的翻译准确性。在猪足孢子虫中，已经鉴定出编码S4和S5的基因。在这里，我们鉴定了编码S12蛋白同源物的基因，并表明它与遗传上已知的AS6基因相同。猪波孢子虫已被用于翻译准确性突变体的密集搜索(Coppin-Raynal et al. 1988)。几个因素参与维护的准确性已确定生物体包括tRNA抑制su4-1和su8-1 (Debuchy et al . 1985年),以及延长因子eEF1A编码由AS4 (Silar et al . 1994),终止因素eRF1和eRF3编码分别由su1和俗/ AS2(方面et al . 1998),核糖体蛋白质S12编码由AS1 (Dequard-Chablat et al . 1994), S7编码由su12 (Silar et al . 1997年)和S1编码,su3 (Silar et al . 2003年)。S12、S7和S1分别是大肠杆菌S4和S5核糖体蛋白同源基因的编码，因此S12、S7和S1是鹅螺旋体对核糖体蛋白的编号(Dequard-Chablat et al. 1986)。这两种蛋白质是在原核生物和真核生物中保存了20多亿年的精确中心的一部分(Alksne et al. 1993)。该中心含有与大肠杆菌S12蛋白相对应的第三种蛋白质，该蛋白质仍有待于在P. anserina中鉴定。这种蛋白是高度保守的，在迄今为止所研究的所有原核生物和真核生物中都是必需的(Alksne et al. 1993)。为了在猪尾丝虫病中鉴定编码该蛋白的基因，我们利用了该真菌的完整基因组序列(可在http://podospora.igmors.u-psud.fr上获得)。我们首先在序列图上找到了S12家族中参与胞质翻译的鸡尾弓形虫蛋白的编码基因。为此，我们使用BLAST搜索了酵母S28蛋白的基因组序列，该蛋白是大肠杆菌S12蛋白的真核同源物(Alksne et al. 1993)。单个CDS, Pa_3_536，获得显著分数(10;图1).它与酿酒酵母s -72 S28蛋白具有89%的同源性和94%的相似性。迄今为止测序的所有生物体的完整基因组中至少存在一个同源物，并且该基因在真菌中高度保守(图1)。例如，鹅尾假丝虫和粗神经孢子虫的蛋白质99%相同。Pa_3_5360位于3号染色体右臂的一个区域，该区域将以70%的二次分裂频率分离，这强烈表明它可能对应于先前已知的AS6基因的CDS，因为(1)AS6位于遗传图谱上的这个位置(Picard-Bennoun et al. 1980)， (2) AS6控制翻译精度(Picard-Bennoun 1981)。(3)该基因的许多突变被恢复为su12突变的抑制因子，因此(Dequard-Chablat 1986); (4) AS6编码一种核糖体蛋白(在鹅尾丝虫命名法中称为S19;Dequard-Chablat et al. 1986)。为了证实这一点，我们首先对AS6-1、AS6-2和AS6-5突变体中存在的三个Pa_3_5360等位基因进行了测序。以寡核苷酸5' ttggagatcccatcaaag -3'和5'- agcctcgcagactcacaca -3'和三个突变体基因组DNA为模板扩增DNA片段。然后进行DNA片段的完整测序，并与野生型进行比较。AS6-1包含一个C到G的转换，将精氨酸73转化为甘氨酸，AS6-2包含一个G到a的转换，将天冬氨酸90转化为天冬氨酸，AS6-5包含一个C到T的转换，将丙氨酸113转化为缬氨酸(图1)。最后，我们将AS6-1突变体与质粒pAS6(质粒GA0AA399BE01来自P。
{"title":"Podospora anserina AS6 gene encodes the cytosolic ribosomal protein of the E. coli S12 family","authors":"M. Déquard-Chablat, P. Silar","doi":"10.4148/1941-4765.1111","DOIUrl":"https://doi.org/10.4148/1941-4765.1111","url":null,"abstract":"The ribosomal proteins of the E. coli S4, S5 and S12 families that are part of the ribosome accuracy center control translation accuracy both in prokaryotes and eukaryotes. In Podospora anserina, genes coding for S4 and S5 have already been identified. Here, we identify the gene coding for the S12 protein homologue and show that it is identical to the genetically known AS6 gene. 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/vol53/iss1/8 26 Fungal Genetics Newsletter Podospora anserina AS6 gene encodes the cytosolic ribosomal protein of the E. coli S12 family Michelle Dequard-Chablat and Philippe Silar 1 1, 2 Institut de Génétique et Microbiologie, Bât. 400 Université de Paris 11, 91405 Orsay cedex, France. 1 UFR de Biochimie, Université de Paris 7 Denis Diderot, case 7006, 2 place Jussieu, 75005, Paris, France 2 To whom correspondence may be addressed: michelle.chablat@igmors.u-psud.fr or philippe.silar@igmors.u-psud.fr Fungal Genetics Newsletter 53: 26-29 The ribosomal proteins of the E. coli S4, S5 and S12 families that are part of the ribosome accuracy center control translation accuracy both in prokaryotes and eukaryotes. In Podospora anserina, genes coding for S4 and S5 have already been identified. Here, we identify the gene coding for the S12 protein homologue and show that it is identical to the genetically known AS6 gene. Podospora anserina has been used in intensive search of translation accuracy mutants (Coppin-Raynal et al. 1988). Several factors involved in the maintenance of accuracy have been identified in this organism including the tRNA suppressors su4-1 and su8-1 (Debuchy et al. 1985), as well as elongation factor eEF1A coded by AS4 (Silar et al. 1994), termination factors eRF1 and eRF3 coded by su1 and su2/AS2 respectively (Gagny et al. 1998), ribosomal proteins S12 coded by AS1 (Dequard-Chablat et al. 1994), S7 coded by su12 (Silar et al. 1997) and S1 coded by su3 (Silar et al. 2003). S12, S7 and S1 refer to the P. anserina numbering for ribosomal proteins (Dequard-Chablat et al. 1986) since the su12 and su3 genes code for the ribosomal proteins homologues of the E. coli S4 and S5, respectively. These two proteins are part of an accuracy center that has been conserved for more than two billion years in both prokaryotes and eukaryotes (Alksne et al. 1993). The center contains a third protein corresponding to the E. coli S12 protein, which remains to be identified in P. anserina. This protein is highly conserved and essential in all prokaryotes and eukaryotes investigated to date (Alksne et al. 1993). To identify the gene coding this protein in P. anserina, we took advantage of the availability of the complete genomic sequence of this fungus (available at http://podospora.igmors.u-psud.fr). We first located on the sequence map the gene encoding the P. anserina protein of the S1","PeriodicalId":12490,"journal":{"name":"Fungal Genetics Reports","volume":"16 1","pages":"26-29"},"PeriodicalIF":0.0,"publicationDate":"2006-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"89877267","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}
Lithium (Li) ions are known to affect Neurospora crassa’s growth speed and circadian clock period, while elevated temperatures abolish these influences. We wondered whether Li has also an effect on conidia size. We used cryo-SEM to investigate this question and report here the results of 1720 measurements showing that at 20°C the long and short conidial axes are significantly reduced at high Li concentrations (10-15 mM), while the ratio between the long and short axes remains approximately constant. An increased temperature (30°C) appears to abolish the Li effect on conidia size. 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/vol53/iss1/10 34 Fungal Genetics Newsletter Influence of Lithium ions on conidiophore size in Neurospora crassa Bodil Aase, Ingunn W. Jolma and Peter Ruoff Department of Mathematics and Natural Science, University of Stavanger, N-4036 Stavanger, Norway corresponding author. email: peter.ruoff@uis.no. # Fungal Genetics Newsletter 53:34-36 Lithium (Li) ions are known to affect Neurospora crassa’s growth speed and circadian clock period, while elevated temperatures abolish these influences. We wondered whether Li has also an effect on conidia size. We used cryo-SEM to investigate this question and report here the results of 1720 measurements showing that at 20°C the long and short conidial axes are significantly reduced at high Li concentrations (10-15 mM), while the ratio between the long and short axes remains approximately constant. An increased temperature (30°C) appears to abolish the Li effect on conidia size. Lithium (Li) has a profound influence on Neurospora crassa’s growth rate and circadian period (Engelmann 1987; Davis 2000; Dunlap and Loros 2004). Typically, at extracellular concentrations of 10 mM LiCl, the growth rate is significantly reduced and the circadian clock begins to get disrupted (Engelmann 1987; Lakin-Thomas 1993; Jolma et al. 2006). Interestingly, increased temperature can abolish the Li effect, possibly by an increased dissociation between Li and its assumed targets (Jolma et al. 2006). Because of the macroscopically distinct differences in conidiation when Neurospora is grown in the presence or absence of Li, we wondered whether there might be also a difference in microscopic conidiation, for example conidia size. In order to answer this question, we performed a study using a Zeiss Supra VP35 scanning electron microscope (SEM) with a Polaron cryo stage. The bd a strain (FGSC #1859) was grown on Petri dishes in LD (12h:12h) at 20°C or 30°C using Vogel’s medium as previously described (Jolma et al. 2006). Li was added to the medium as LiCl. Samples were taken from Petri dishes that showed approximately the same amount of total growth (=growth speed X growth time). Because of the dependence of the speed of this organism’s growth on temperature and LiCl
众所周知，锂离子会影响粗神经孢子虫的生长速度和生物钟周期，而升高的温度会消除这些影响。我们想知道Li是否也对分生孢子的大小有影响。我们使用冷冻扫描电镜(cro - sem)研究了这个问题，并在这里报告了1720年的测量结果，结果表明，在20°C时，在高Li浓度(10-15 mM)下，分生孢子的长轴和短轴显著减少，而长轴和短轴之间的比例保持大致不变。温度升高(30℃)似乎消除了Li对分生孢子大小的影响。本作品采用知识共享署名-相同方式共享4.0许可协议。这篇论文发表在《真菌遗传学报告:http://newprairiepress.org/fgr/vol53/iss1/10 34真菌遗传学通讯》上，锂离子对粗神经孢子虫Bodil Aase分生孢子大小的影响，Ingunn W. Jolma和Peter Ruoff，斯塔万格大学数学与自然科学系，N-4036斯塔万格，挪威。电子邮件:peter.ruoff@uis.no。#真菌遗传学通讯53:34-36已知锂离子会影响粗神经孢子虫的生长速度和生物钟周期，而升高的温度会消除这些影响。我们想知道Li是否也对分生孢子的大小有影响。我们使用冷冻扫描电镜(cro - sem)研究了这个问题，并在这里报告了1720年的测量结果，结果表明，在20°C时，在高Li浓度(10-15 mM)下，分生孢子的长轴和短轴显著减少，而长轴和短轴之间的比例保持大致不变。温度升高(30℃)似乎消除了Li对分生孢子大小的影响。锂(Li)对粗神经孢子虫(Neurospora crassa)的生长速度和昼夜节律有深远的影响(Engelmann 1987;戴维斯2000;Dunlap and Loros 2004)。通常，在细胞外浓度为10 mM LiCl时，生长速度显著降低，生物钟开始被打乱(Engelmann 1987;Lakin-Thomas 1993;Jolma et al. 2006)。有趣的是，升高的温度可以消除锂效应，可能是由于锂与其假设目标之间的解离增加(Jolma et al. 2006)。由于神经孢子在Li存在或不存在的情况下生长时，在宏观上存在明显的分生差异，我们想知道在微观上是否也存在分生差异，例如分生孢子的大小。为了回答这个问题，我们使用带有极化子冷冻级的蔡司Supra VP35扫描电子显微镜(SEM)进行了研究。bd a菌株(fgsc# 1859)在20°C或30°C的培养皿中培养(12h:12h)，使用Vogel培养基，如前所述(Jolma et al. 2006)。Li以LiCl的形式加入到培养基中。样品取自培养皿，培养皿显示出大致相同的总生长量(=生长速度X生长时间)。由于该生物的生长速度依赖于温度和LiCl浓度(Jolma et al. 2006)，因此采样前的生长时间变化如表1所示。表1:生长时间前期分析T，°C LiCl, mM GT，天20 0 3 20 5 3 20 10 10 10 10 15 10 30 10 1 T:温度;LiCl: LiCl浓度;GT:生长时间分生孢子大小分别为长轴和短轴l和s。图为分生孢子在20°C、15 mM Li条件下生长。用软木钻覆盖生长区域并在生长区域后面约5毫米处切割样品。切下的样品被放在Al-stub上，其表面有胶状石墨。将胶合后的样品快速冷冻在氮泥中，然后转移到预冷SEM阶段。然后用金钯合金涂覆样品80秒。在SEM分析中，使用了显微镜的“In- lens”检测器。分生孢子呈椭球状，分生孢子的长轴l和短轴s(图1)可直接在样品上测定，作为分生孢子大小的近似度量。新草原出版社2017年出版
{"title":"Influence of Lithium ions on conidiophore size in Neurospora crassa","authors":"B. Aase, I. W. Jolma, P. Ruoff","doi":"10.4148/1941-4765.1113","DOIUrl":"https://doi.org/10.4148/1941-4765.1113","url":null,"abstract":"Lithium (Li) ions are known to affect Neurospora crassa’s growth speed and circadian clock period, while elevated temperatures abolish these influences. We wondered whether Li has also an effect on conidia size. We used cryo-SEM to investigate this question and report here the results of 1720 measurements showing that at 20°C the long and short conidial axes are significantly reduced at high Li concentrations (10-15 mM), while the ratio between the long and short axes remains approximately constant. An increased temperature (30°C) appears to abolish the Li effect on conidia size. 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/vol53/iss1/10 34 Fungal Genetics Newsletter Influence of Lithium ions on conidiophore size in Neurospora crassa Bodil Aase, Ingunn W. Jolma and Peter Ruoff Department of Mathematics and Natural Science, University of Stavanger, N-4036 Stavanger, Norway corresponding author. email: peter.ruoff@uis.no. # Fungal Genetics Newsletter 53:34-36 Lithium (Li) ions are known to affect Neurospora crassa’s growth speed and circadian clock period, while elevated temperatures abolish these influences. We wondered whether Li has also an effect on conidia size. We used cryo-SEM to investigate this question and report here the results of 1720 measurements showing that at 20°C the long and short conidial axes are significantly reduced at high Li concentrations (10-15 mM), while the ratio between the long and short axes remains approximately constant. An increased temperature (30°C) appears to abolish the Li effect on conidia size. Lithium (Li) has a profound influence on Neurospora crassa’s growth rate and circadian period (Engelmann 1987; Davis 2000; Dunlap and Loros 2004). Typically, at extracellular concentrations of 10 mM LiCl, the growth rate is significantly reduced and the circadian clock begins to get disrupted (Engelmann 1987; Lakin-Thomas 1993; Jolma et al. 2006). Interestingly, increased temperature can abolish the Li effect, possibly by an increased dissociation between Li and its assumed targets (Jolma et al. 2006). Because of the macroscopically distinct differences in conidiation when Neurospora is grown in the presence or absence of Li, we wondered whether there might be also a difference in microscopic conidiation, for example conidia size. In order to answer this question, we performed a study using a Zeiss Supra VP35 scanning electron microscope (SEM) with a Polaron cryo stage. The bd a strain (FGSC #1859) was grown on Petri dishes in LD (12h:12h) at 20°C or 30°C using Vogel’s medium as previously described (Jolma et al. 2006). Li was added to the medium as LiCl. Samples were taken from Petri dishes that showed approximately the same amount of total growth (=growth speed X growth time). Because of the dependence of the speed of this organism’s growth on temperature and LiCl","PeriodicalId":12490,"journal":{"name":"Fungal Genetics Reports","volume":"29 1","pages":"34-36"},"PeriodicalIF":0.0,"publicationDate":"2006-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"78227122","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}
Aspergillus nidulans grown under standard laboratory conditions does not show circadian rhythmic growth. The presence of a circadian clock was demonstrated in A. nidulans at the level of gene expression (Greene et al. 2003), but a visually observable rhythm is lacking. We recently observed a remarkable rhythmic growth pattern in the formation of conidiospores and ascospores in a fludioxonil resistant mutant of A. nidulans (fldA1) grown in a dark incubator. This is reminiscent of a circadian rhythm. We found however that our observed rhythm is induced by light (leaking into the ‘dark’ incubator) and is not self-sustainable. In absolute darkness or constant light the rhythm is lost; therefore, we conclude that the rhythm is not a true intrinsic circadian rhythm.
在标准实验室条件下生长的中性曲霉不显示昼夜节律生长。在基因表达水平上，毛竹中存在生物钟(Greene et al. 2003)，但缺乏视觉上可观察到的节律。我们最近观察到在黑暗培养箱中培养的一株抗氟虫腈突变株(fldA1)在分孢子孢子和子囊孢子的形成过程中出现了显著的节律性生长模式。这让人想起昼夜节律。然而，我们发现我们观察到的节奏是由光引起的(泄漏到“黑暗”孵化器中)，并不是自我持续的。在绝对的黑暗或恒定的光照下，节奏丧失了;因此，我们得出结论，该节律不是真正的内在昼夜节律。
{"title":"Non-circadian light inducible rhythm in Aspergillus nidulans.","authors":"S. Schoustra","doi":"10.4148/1941-4765.1110","DOIUrl":"https://doi.org/10.4148/1941-4765.1110","url":null,"abstract":"Aspergillus nidulans grown under standard laboratory conditions does not show circadian rhythmic growth. The presence of a circadian clock was demonstrated in A. nidulans at the level of gene expression (Greene et al. 2003), but a visually observable rhythm is lacking. We recently observed a remarkable rhythmic growth pattern in the formation of conidiospores and ascospores in a fludioxonil resistant mutant of A. nidulans (fldA1) grown in a dark incubator. This is reminiscent of a circadian rhythm. We found however that our observed rhythm is induced by light (leaking into the ‘dark’ incubator) and is not self-sustainable. In absolute darkness or constant light the rhythm is lost; therefore, we conclude that the rhythm is not a true intrinsic circadian rhythm.","PeriodicalId":12490,"journal":{"name":"Fungal Genetics Reports","volume":"68 1","pages":"23-25"},"PeriodicalIF":0.0,"publicationDate":"2006-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"82389201","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}
Mating type tests in Neurospora crassa are an important way to characterize strains. Since most of the knock-out mutants developed as part of the functional genomics program (Colot et al., 2006) have little obvious phenotype we have undertaken to test the mating type of all of the knock-strains that are sent to the FGSC. Our original mating type test protocol is similar to that described by Smith (1962) and involves growing a lawn of the fluffy tester strains (4317 or 4347) on cornmeal agar (Difco) in 15 cm petri plates. This is also described in the online Neurospora protocol, "How to use fluffy testers for determining mating type and for other applications" (http://www.fgsc.net/Neurospora/NeurosporaProtocolGuide.htm). The strains to be tested are grown on Vogels minimal (Vogel, 1956) or appropriately supplemented medium (McCluskey, 2003) and small amounts of conidia are transferred to a spot on a grid. Using this technique, thirty to forty strains can be tested on two plates. While robust, this technique is labor intensive and because the plate is opened for each inoculation there is the possibility that occasional stray conidia could confound the results.
粗神经孢子虫的交配型试验是鉴定菌株的重要方法。由于作为功能基因组学计划的一部分(Colot et al.， 2006)开发的大多数敲除突变体几乎没有明显的表型，因此我们开始测试发送给FGSC的所有敲除菌株的交配类型。我们最初的交配型试验方案与Smith(1962)所描述的相似，包括在玉米粉琼脂(Difco)上在15厘米的培养皿中种植蓬松测试菌株(4317或4347)。在线Neurospora协议中也有描述，“如何使用蓬松的测试器来确定交配类型和其他应用”(http://www.fgsc.net/Neurospora/NeurosporaProtocolGuide.htm)。待测试菌株在Vogels minimal (Vogel, 1956)或适当补充的培养基(McCluskey, 2003)上生长，少量分生孢子被转移到网格上的一个点上。使用这种技术，可以在两个板上测试30到40个应变。虽然稳健，但这种技术是劳动密集型的，因为每次接种都要打开培养皿，偶尔的散生孢子可能会混淆结果。
{"title":"High throughput mating tests in Neurospora crassa","authors":"K. McCluskey, Rachel L. Yedlin, S. Walker","doi":"10.4148/1941-4765.1109","DOIUrl":"https://doi.org/10.4148/1941-4765.1109","url":null,"abstract":"Mating type tests in Neurospora crassa are an important way to characterize strains. Since most of the knock-out mutants developed as part of the functional genomics program (Colot et al., 2006) have little obvious phenotype we have undertaken to test the mating type of all of the knock-strains that are sent to the FGSC. Our original mating type test protocol is similar to that described by Smith (1962) and involves growing a lawn of the fluffy tester strains (4317 or 4347) on cornmeal agar (Difco) in 15 cm petri plates. This is also described in the online Neurospora protocol, \"How to use fluffy testers for determining mating type and for other applications\" (http://www.fgsc.net/Neurospora/NeurosporaProtocolGuide.htm). The strains to be tested are grown on Vogels minimal (Vogel, 1956) or appropriately supplemented medium (McCluskey, 2003) and small amounts of conidia are transferred to a spot on a grid. Using this technique, thirty to forty strains can be tested on two plates. While robust, this technique is labor intensive and because the plate is opened for each inoculation there is the possibility that occasional stray conidia could confound the results.","PeriodicalId":12490,"journal":{"name":"Fungal Genetics Reports","volume":"6 1","pages":"20-22"},"PeriodicalIF":0.0,"publicationDate":"2006-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"80352944","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}
Strains of Neurospora crassa exist as two alternative mating type forms, A and a; differences in mating type are required for the initiation of the sexual cycle (Shiu and Glass, 2000). The mating-type (mat) locus also acts as a heterokaryon incompatibility (het) locus, such that hyphal fusion between A and a strains results in a heterokaryon that shows extremely inhibited growth, absence of conidiation, and hyphal compartmentation and death (Glass et al., 2000). The A and a mating type sequences occupy the same locus in A and a strains, but are highly dissimilar in sequence. The mat a-1 gene, which encodes a putative HMG (high mobility group) type of transcriptional regulator, provides all the functions for the a mating type, including mating, ascospore formation, and heterokaryon incompatibility (Chang and Staben, 1994). The mat A locus encodes three proteins. MAT A-2 and MAT A-3 are responsible for ascospore formation (Ferreira et al., 1998); MAT A-3 is a putative HMG type of transcriptional regulator. MAT A-1 is predicted to be a a-domain type of transcriptional regulator and is both necessary and sufficient to confer A mating specificity and trigger heterokaryon incompatibility with a strains (Glass et al., 1990). Mutations in an unlinked locus, tol, suppress mating-type incompatibility such that tol A and tol a strains are capable of forming a vigorous heterokaryon (Newmeyer, 1970; Shiu and Glass, 1999).
粗神经孢子虫以A和A两种交配型存在;交配类型的差异是性周期开始所必需的(Shiu和Glass, 2000)。交配型位点(mat)也起到异核不相容位点(het)的作用，因此a株和a株之间的菌丝融合会导致异核体的生长受到极大抑制，菌丝分离和死亡(Glass et al.， 2000)。A和A交配型序列在A和A株中占有相同的位点，但在序列上有很大的差异。mat a-1基因编码一种假定的HMG(高迁移率组)型转录调节因子，为a交配型提供所有功能，包括交配、子囊孢子形成和异核体不相容(Chang和Staben, 1994)。mat A基因座编码三种蛋白质。MAT A-2和MAT A-3负责子囊孢子的形成(Ferreira et al.， 1998);MAT a -3是一种假定的HMG型转录调节因子。据预测，MAT a- 1是a结构域类型的转录调节因子，是赋予a交配特异性和触发菌株异核不相容的必要和充分条件(Glass et al.， 1990)。非连锁位点tol的突变抑制了交配型不亲和性，因此tol A和tol A菌株能够形成强有力的异核子(Newmeyer, 1970;Shiu and Glass, 1999)。
{"title":"Sequences important for heterokaryon incompatibility function in MAT A-1 of Neurospora crassa","authors":"P. Shiu, N. Glass","doi":"10.4148/1941-4765.1108","DOIUrl":"https://doi.org/10.4148/1941-4765.1108","url":null,"abstract":"Strains of Neurospora crassa exist as two alternative mating type forms, A and a; differences in mating type are required for the initiation of the sexual cycle (Shiu and Glass, 2000). The mating-type (mat) locus also acts as a heterokaryon incompatibility (het) locus, such that hyphal fusion between A and a strains results in a heterokaryon that shows extremely inhibited growth, absence of conidiation, and hyphal compartmentation and death (Glass et al., 2000). The A and a mating type sequences occupy the same locus in A and a strains, but are highly dissimilar in sequence. The mat a-1 gene, which encodes a putative HMG (high mobility group) type of transcriptional regulator, provides all the functions for the a mating type, including mating, ascospore formation, and heterokaryon incompatibility (Chang and Staben, 1994). The mat A locus encodes three proteins. MAT A-2 and MAT A-3 are responsible for ascospore formation (Ferreira et al., 1998); MAT A-3 is a putative HMG type of transcriptional regulator. MAT A-1 is predicted to be a a-domain type of transcriptional regulator and is both necessary and sufficient to confer A mating specificity and trigger heterokaryon incompatibility with a strains (Glass et al., 1990). Mutations in an unlinked locus, tol, suppress mating-type incompatibility such that tol A and tol a strains are capable of forming a vigorous heterokaryon (Newmeyer, 1970; Shiu and Glass, 1999).","PeriodicalId":12490,"journal":{"name":"Fungal Genetics Reports","volume":"24 1","pages":"15-19"},"PeriodicalIF":0.0,"publicationDate":"2006-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"82556570","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}
Often one needs to determine a suitable concentration of a previously untested nutrient or inhibitor to use in subsequent experiments. Creative Commons License This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. This brief note is available in Fungal Genetics Reports: http://newprairiepress.org/fgr/vol53/iss1/11
{"title":"A simple way to make a dilution series","authors":"R. L. Metzenberg","doi":"10.4148/1941-4765.1114","DOIUrl":"https://doi.org/10.4148/1941-4765.1114","url":null,"abstract":"Often one needs to determine a suitable concentration of a previously untested nutrient or inhibitor to use in subsequent experiments. Creative Commons License This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. This brief note is available in Fungal Genetics Reports: http://newprairiepress.org/fgr/vol53/iss1/11","PeriodicalId":12490,"journal":{"name":"Fungal Genetics Reports","volume":"33 1","pages":"37"},"PeriodicalIF":0.0,"publicationDate":"2006-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"82581840","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}
An analysis of the genome of Neurospora crassa has identified genes encoding 84 putative glycosyl hydrolases, representing 24 different families in Henrissat's classification. Functionally, they include enzymes for the degradation of all major polysaccharides (including cellulase, hemicellulase, chitinase and pectinase).There is evidence of high levels of gene amplification, despite the presence of RIP, as there are eight representatives of family 3 (beta-glucosidases and xylosidases), five of family 7 (endoand exo-glucanases), six of family 13 (amylases and maltase), nine of family 18 (chitinase), eight of family 47 (ER alpha-mannosidases), eleven of family 61 (endoglucanases) and seven of family 76 (alpha-mannanases). 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/vol53/iss1/4
{"title":"Glycosyl Hydrolase Genes and Enzymes of Neurospora crassa","authors":"A. Radford","doi":"10.4148/1941-4765.1107","DOIUrl":"https://doi.org/10.4148/1941-4765.1107","url":null,"abstract":"An analysis of the genome of Neurospora crassa has identified genes encoding 84 putative glycosyl hydrolases, representing 24 different families in Henrissat's classification. Functionally, they include enzymes for the degradation of all major polysaccharides (including cellulase, hemicellulase, chitinase and pectinase).There is evidence of high levels of gene amplification, despite the presence of RIP, as there are eight representatives of family 3 (beta-glucosidases and xylosidases), five of family 7 (endoand exo-glucanases), six of family 13 (amylases and maltase), nine of family 18 (chitinase), eight of family 47 (ER alpha-mannosidases), eleven of family 61 (endoglucanases) and seven of family 76 (alpha-mannanases). 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/vol53/iss1/4","PeriodicalId":12490,"journal":{"name":"Fungal Genetics Reports","volume":"3 1","pages":"12-14"},"PeriodicalIF":0.0,"publicationDate":"2006-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"72825098","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}
To better characterize handling parameters for the arrayed mutants prepared for the Neurospora functional genomics program, we have put 7 day old conidia from strain FGSC 2489 through a series of cycles of freezing at -80 C in 25% glycerol and 3.5% reconstituted non-fat dry milk. Creative Commons License This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. This brief note is available in Fungal Genetics Reports: http://newprairiepress.org/fgr/vol53/iss1/12
{"title":"The effect of repeated freeze-thaw cycles on cryopreserved Neurospora crassa samples.","authors":"K. McCluskey, A. Wiest, S. Walker","doi":"10.4148/1941-4765.1115","DOIUrl":"https://doi.org/10.4148/1941-4765.1115","url":null,"abstract":"To better characterize handling parameters for the arrayed mutants prepared for the Neurospora functional genomics program, we have put 7 day old conidia from strain FGSC 2489 through a series of cycles of freezing at -80 C in 25% glycerol and 3.5% reconstituted non-fat dry milk. Creative Commons License This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. This brief note is available in Fungal Genetics Reports: http://newprairiepress.org/fgr/vol53/iss1/12","PeriodicalId":12490,"journal":{"name":"Fungal Genetics Reports","volume":"84 1","pages":"37"},"PeriodicalIF":0.0,"publicationDate":"2006-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"86853973","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}
Kaitlyn Beasley, T. Lamb, W. Versaw, Deborah Bell-Pedersen
We describe the construction of a Neurospora crassa Mauriceville strain carrying the ras-1bd mutation marked by the bacterial hygromycin resistance gene, hph (new FGSC # 10156). This strain is valuable for mapping mutations in Oak Ridge strains that carry the bd mutation. 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/vol53/iss1/9 30 Fungal Genetics Newsletter A ras-1 Mauriceville strain for mapping mutations in Oak Ridge ras-1 strains bd bd A. Kaitlyn Beasley*, Teresa M. Lamb*, Wayne K. Versaw and Deborah Bell-Pedersen* *Center for Biological Clocks Research and Department of Biology, Texas A&M University, College Station, TX 77843 Fungal Genetics Newsletter 53:30-33 We describe the construction of a Neurospora crassa Mauriceville strain carrying the ras-1 mutation marked by the bacterial bd hygromycin resistance gene, hph (new FGSC # 10156). This strain is valuable for mapping mutations in Oak Ridge strains that carry the bd mutation. The bd mutation has been a benefit to circadian rhythm research as it slows down wild type growth rates and sharpens the conidial banding pattern on racetubes (Sargent et al., 1966; Bell-Pedersen et al., 2005). Recent work (Belden et al., 2006) has shown that the bd mutation lies in the ras-1 gene. Mutations that affect circadian banding patterns are typically isolated in strains carrying the ras-1 allele. Mapping such mutations by the CAPS method (Jin et al., in press) requires that the mutant phenotype be followed in bd a cross to a wild type Mauriceville strain. Assuming non-linkage, only half of the progeny will be bd, and thus readily scorable. Insertion of the ras-1 allele into the Mauriceville parent renders all progeny useful for segregation analysis. Furthermore, bd marking ras-1 with the hygromycin resistance gene, hph, permits a simple drug resistance test to determine linkage to ras-1. bd The strategy for gene replacement was similar to published split marker gene deletion strategies (Catlett et al., 2003; Colot et al., 2006). However, rather than deleting the wild type gene, we replaced it with the bd allele and inserted the hph gene in the 3'UTR at position + 225 from the stop codon (Figure 1). Figure 1. Schematic diagram of ras-1 genetic manipulations. Regions of homology are indicated by vertical hatch marks; there are 763 bp of homology between the split hph fragments, 3223 bp of homology at the 5' end, and 2582 bp of homology at the 3' end. Pvu II and Xho I sites are marked to show the differences between the wild type (wt) genomic (g) locus and the proper ras1 -hph integrant. The dashed line indicates a gap where the hph gene is integrated and the dotted lines indicate flanking genomic bd DNA that remains unchanged. The replacement fragments were generated in two steps. The first step involved amplification of ras-1 genomic regions from the bd O
我们描述了一株携带细菌耐潮霉素基因hph(新fgsc# 10156)标记的ras-1bd突变的粗糙神经孢子虫Mauriceville菌株的构建。该菌株对绘制携带bd突变的橡树岭菌株的突变图谱很有价值。本作品采用知识共享署名-相同方式共享4.0许可协议。这篇常规论文发表在《真菌遗传学报告》上:http://newprairiepress.org/fgr/vol53/iss1/9 30真菌遗传学通讯A. Kaitlyn Beasley*， Teresa M. Lamb*， Wayne K. Versaw, Deborah Bell-Pedersen* *德州农工大学生物时钟研究中心，大学城我们描述了一株携带细菌耐水霉素基因hph(新fgsc# 10156)标记的ras-1突变的草神经孢子虫Mauriceville菌株的构建。该菌株对绘制携带bd突变的橡树岭菌株的突变图谱很有价值。bd突变对昼夜节律研究是有益的，因为它减缓了野生型的生长速度，并使总状管上的分生孢子带型变尖(Sargent et al.， 1966;Bell-Pedersen et al.， 2005)。最近的研究(Belden et al.， 2006)表明，bd突变存在于ras-1基因中。影响昼夜带带模式的突变通常在携带ras-1等位基因的菌株中分离出来。用CAPS方法绘制这种突变图谱(Jin et al.， in press)需要在与野生型Mauriceville菌株杂交时遵循突变表型。假设非连锁，只有一半的后代将是bd，因此很容易得分。将ras-1等位基因插入到毛里斯维尔亲本中，使所有后代对分离分析都有用。此外，bd用潮霉素耐药基因hph标记ras-1，允许进行简单的耐药试验以确定与ras-1的连锁关系。bd基因替换策略类似于已发表的分裂标记基因删除策略(Catlett et al.， 2003;Colot et al.， 2006)。然而，我们没有删除野生型基因，而是用bd等位基因替换它，并在停止密码子+ 225位置的3'UTR中插入hph基因(图1)。ras-1基因操作示意图。同源区域用垂直的舱口标记表示;HPH片段的同源性为763 bp，其中5′端同源性为3223 bp, 3′端同源性为2582 bp。Pvu II和Xho I位点被标记，以显示野生型(wt)基因组(g)位点与适当的ras1 -hph整合子之间的差异。虚线表示hph基因整合的间隙，虚线表示两侧基因组bd DNA保持不变。替换片段分两步生成。第一步是扩增橡树岭bd菌株(FGSC 1858)的ras-1基因组区域。用引物ras1 F1和ras1-hyg R1扩增5′区，用引物hyg-ras1 F2和ras1 R2扩增3′区。第二步是产生含有一部分细菌hph基因融合到ras-1的5'或3'区域的杂交片段(图1)。5'分裂标记片段是通过扩增ras-1的5'区域和完整的hph基因产生的，引物为ras1 F1和YG2。用引物HY2和ras1 R2扩增ras-1的3′区和完整的hph基因，得到3′分裂标记片段bd。用引物M13F2和M13R2扩增pBP15 (D. Ebbole赠送)，获得完整的hph基因bd。pBP15质粒含有来自pCB1003 (Carroll et al.， 1994)亚克隆到pBluescript II SK的EcoRV位点的1.4kb HpaI PtrpC-hph片段(New Prairie Press, 2017)
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