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Genomic organization of the Neurospora crassa gsn gene: possible involvement of the STRE and HSE elements in the modulation of transcription during heat shock

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Abstract

Glycogen synthase, an enzyme involved in glycogen biosynthesis, is regulated by phosphorylation and by the allosteric ligand glucose-6-phosphate (G6P). In addition, enzyme levels can be regulated by changes in gene expression. We recently cloned a cDNA for glycogen synthase ( gsn) from Neurospora crassa, and showed that gsn transcription decreased when cells were exposed to heat shock (shifted from 30°C to 45°C). In order to understand the mechanisms that control gsn expression, we isolated the gene, including its 5′ and 3′ flanking regions, from the genome of N. crassa. An ORF of approximately 2.4 kb was identified, which is interrupted by four small introns (II–V). Intron I (482 bp) is located in the 5′UTR region. Three putative Transcription Initiation Sites (TISs) were mapped, one of which lies downstream of a canonical TATA-box sequence (5′-TGTATAAA-3′). Analysis of the 5′-flanking region revealed the presence of putative transcription factor-binding sites, including Heat Shock Elements (HSEs) and STress Responsive Elements (STREs). The possible involvement of these motifs in the negative regulation of gsn transcription was investigated using Electrophoretic Mobility Shift Assays (EMSA) with nuclear extracts of N. crassa mycelium obtained before and after heat shock, and DNA fragments encompassing HSE and STRE elements from the 5′-flanking region. While elements within the promoter region are involved in transcription under heat shock, elements in the 5′UTR intron may participate in transcription during vegetative growth. The results thus suggest that N. crassa possesses trans -acting elements that interact with the 5′-flanking region to regulate gsn transcription during heat shock and vegetative growth.

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Acknowledgements

We thank Dr. H. F. Terenzi (Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, USP), Dr. R. M. de Paula, and Dr. A. M. Silva (Instituto de Química-USP, São Paulo) for suggestions and discussion of this work; Dr. N. Monesi, Dr. M. Vallim, Dr. J. C. Oliveira, and Dr. A. O. B. Ribon for helpful suggestions on primer extension assay and EMSA. F.Z.F. was supported by a fellowship from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). This work was supported by grants from FAPESP to M.C.B. The work was carried out in compliance with the current laws governing genetic experimentation in Brazil

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  1. Departamento de Bioquímica e Tecnologia Química, Instituto de Química, UNESP, 14800-900, Araraquara, SP, Brazil

    F. Zanolli Freitas & M. C. Bertolini

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  1. F. Zanolli Freitas
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  2. M. C. Bertolini
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Correspondence to M. C. Bertolini.

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Communicated by M. Collart

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Zanolli Freitas, F., Bertolini, M.C. Genomic organization of the Neurospora crassa gsn gene: possible involvement of the STRE and HSE elements in the modulation of transcription during heat shock. Mol Genet Genomics 272, 550–561 (2004). https://doi.org/10.1007/s00438-004-1086-5

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  • DOI: https://doi.org/10.1007/s00438-004-1086-5

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