Synchronization of DNA synthesis in Neurospora crassa by 2?-deoxyadenosine and spore selection

Fletcher, Margaret H.; Trinci, Anthony P. J.

doi:10.1007/bf00422314

Cited by 3 publications

(1 citation statement)

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“…7A). While picolinic acid, which rapidly slows DNA accumulation (26), and deoxyadenosine, a DNA synthesis inhibitor (13), have been used in the past to synchronize the nuclear division cycle in N. crassa, we suggest that shifting Neurospora from high to low temperature or supplementation with spermidine is less invasive than the former treatments. To that end, we are currently defining the precise growth conditions to synchronize the cell cycle of spe-1 ndc strains with the goal of generating a system that will allow synchronized DNA synthesis upon addition of spermidine.…”

Section: Discussionmentioning

confidence: 99%

Bulk Segregant Analysis Followed by High-Throughput Sequencing Reveals the Neurospora Cell Cycle Gene, ndc-1 , To Be Allelic with the Gene for Ornithine Decarboxylase, spe-1

2011

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With the advent of high-throughput DNA sequencing, it is now straightforward and inexpensive to generate high-density small nucleotide polymorphism (SNP) maps. Here we combined high-throughput sequencing with bulk segregant analysis to expedite mutation mapping. The general map location of a mutation can be identified by a single backcross to a strain enriched in SNPs compared to a standard wild-type strain. Bulk segregant analysis simultaneously increases the likelihood of determining the precise nature of the mutation. We present here a high-density SNP map between Neurospora crassa Mauriceville-1-c (FGSC2225) and OR74A (FGSC2489), the strains most typically used by Neurospora researchers to carry out mapping crosses. We further have demonstrated the utility of the Mauriceville sequence and our approach by mapping the mutation responsible for the only existing temperature-sensitive (ts) cell cycle mutation in Neurospora, nuclear division cycle-1 (ndc-1). The single T-to-C point mutation maps to the gene encoding ornithine decarboxylase (ODC), spe-1 (NCU01271), and changes a Phe to a Ser residue within a highly conserved motif next to the catalytic site of the enzyme. By growth on spermidine and complementation with a wild-type spe-1 gene, we showed that the defect in spe-1 is responsible for the ts ndc-1 mutation. Based on our results, we propose changing ndc-1 to spe-1 ndc , which reflects that this mutation results in an ODC with a specific nuclear division defect.Single nucleotide polymorphism (SNP) maps between organisms with different genetic backgrounds are useful for identifying point mutations that result in an observable phenotype. By mating a mutant with a strain of a different genetic background and then selecting progeny with and without a phenotype, one is able to map the underlying mutation(s) by bulk segregant analysis (29). After undergoing recombination, each progeny exhibiting a specific phenotype will have the same genetic background as the mutant parent in the vicinity of the mutation but a mixture of DNA inherited from either parent in regions of the genome that are unlinked to the mutation. By analyzing DNA from a mixture of phenotypically mutant progeny from the cross, it is possible to define the genetic region containing the mutation and even to identify the mutation itself. This is what is meant by "bulk segregant analysis."Most recently, molecular mapping strategies based on restriction fragment length polymorphisms (RFLP) (28), cleaved amplified polymorphic sequences (CAPS) (20, 22), or microarray-based restriction-site-associated DNA mapping (RAD mapping) (1, 23) have been used to map mutations in Neurospora crassa. These methods can be time-consuming and laborious and therefore expensive. In addition, once the mutation is mapped to a specific region of the genome, additional primer pairs for CAPS mapping need to be ordered and additional SNPs identified to more precisely map the mutation. Typically, researchers will then attempt complementation of phenotypes by candidate genes. On...

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Section: Discussionmentioning

confidence: 99%

Bulk Segregant Analysis Followed by High-Throughput Sequencing Reveals the Neurospora Cell Cycle Gene, ndc-1 , To Be Allelic with the Gene for Ornithine Decarboxylase, spe-1

2011

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An antialgal compound produced by Streptomyces jiujiangensis JXJ 0074T

Zhang

Chen

et al. 2015

Appl Microbiol Biotechnol

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Previous investigations suggested that Streptomyces jiujiangensis JXJ 0074(T) can secrete antialgal compounds. In this study, an antialgal compound was isolated from the cultured broth of S. jiujiangensis JXJ 0074(T) by using bioassay methods. Based on spectroscopic data, the active compound was identified as 2'-deoxyadenosine, which exhibited a greater antialgal activity against cyanobacteria than its analogues such as adenosine, guanosine, and 2'-deoxyguanosine. The antialgal activity of 2'-deoxyadenosine increased with the content and time. 2'-Deoxyadenosine severely damaged the vegetative cells of cyanobacteria, causing crumpling, collapse, expanding, perforation, breakage of filamentous cyanobacteria, and decrease of the chlorophyll. However, 2'-deoxyadenosine seemed to have less impact on the morphology of heterocysts of filamentous cyanobacteria. The superoxide dismutase (SOD) activity in the treated cells of M. aeruginosa FACHB-905 initially increased with 31.14 ± 2.00% higher than that of the control after 36 h and then decreased quickly. On the same time, there were rapid increases in superoxide anion radical (O2 (-)) and malondialdehyde (MDA) contents with 315.53 ± 12.81 and 84.72 ± 6.15% higher than these of the controls at 60 h, respectively. The intracellular microcystin-LR (MC-LR) content in the treated cells of M. aeruginosa FACHB-905 increased by 36.34 ± 7.35% 1 day later, followed by a rapid decrease with a rate of 90.50 ± 1.08% 8 days later, while the extracellular MC-LR content showed no significant difference with the control. Five days after adding 15 μg/ml of 2'-deoxyadenosine to the culture of M. aeruginosa FACHB-905, there was no 2'-deoxyadenosine detected by HPLC, suggesting that 2'-deoxyadenosine completely degraded. This study provides a new clue to screen natural-based antialgal compounds from nucleoside analogues.

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Timing of nuclear division cycle in Neurospora Crassa

Martegani

Tomé²,

Trezzi³

1981

Journal of Cell Science

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The timing of the nuclear division cycle in Neurospora crassa was studied by inhibiting DNA synthesis with hydroxyurea added to exponentially growing cultures at concentrations that do not inhibit cytoplasmic growth. After the addition of hydroxyurea only the nuclei that have passed the S-phase divide, and therefore by counting the increase in the average number of nuclei per hypha, the stage in the cycle in which completion of S phase occurred was determined in different nutritional conditions of exponential growth. The stage at which DNA replication terminates is different in different conditions of exponential growth; however, when the length of G2+M phases is calculated a constancy of these phases is shown. Our data indicate that in N. crassa the coordination between cytoplasmic growth and the nuclear division cycle is achieved mainly through a growth-rate-dependent expansion of the G1 phase, according to a sizer-plus-timer model of control of the nuclear division cycle.

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Synchronization of DNA synthesis in Neurospora crassa by 2?-deoxyadenosine and spore selection

Cited by 3 publications

References 20 publications

Bulk Segregant Analysis Followed by High-Throughput Sequencing Reveals the Neurospora Cell Cycle Gene, ndc-1 , To Be Allelic with the Gene for Ornithine Decarboxylase, spe-1

Bulk Segregant Analysis Followed by High-Throughput Sequencing Reveals the Neurospora Cell Cycle Gene, ndc-1 , To Be Allelic with the Gene for Ornithine Decarboxylase, spe-1

An antialgal compound produced by Streptomyces jiujiangensis JXJ 0074T

Timing of nuclear division cycle in Neurospora Crassa

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