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Genome-wide identification of targets for the archaeal heat shock regulator phr by cell-free transcription of genomic DNA.

ABSTRACT: The hyperthermophilic archaeon Pyrococcus furiosus grows optimally near 100 degrees C and undergoes a heat shock response at 105 degrees C, mediated at least in part by the heat shock regulator Phr. Genes encoding a small heat shock protein (HSP20) and a member of the AAA(+) ATPase are the only known targets of the regulator, but a genetic mutant of Phr has yet to be characterized. We describe here an alternative approach for the identification of the regulon of Phr based on cell-free transcription of fragmented chromosomal DNA in the presence or absence of the regulator and hybridization of in vitro RNA to P. furiosus whole-genome microarrays. Our results confirmed the phr, the hsp20, and the aaa(+) ATPase genes as targets of Phr and also identified six additional open reading frames, PF0624, PF1042, PF1291, PF1292, PF1488, and PF1616, as Phr-responsive genes, which include that encoding di-myo-inositol phosphate synthase. Transcription of the identified novel genes was inhibited by Phr in standard transcription assays, and the novel consensus sequence 5'-TTTAnnnACnnnnnGTnAnnAAAA-3' (uppercase letters denote a high conservation of the bases) was inferred from our data as the Phr recognition motif. Mutational evidence for the significance of this sequence as Phr recognition was provided in DNA-binding experiments.

SUBMITTER: Keese AM

PROVIDER: S-EPMC2820856 | biostudies-other | 2010 Mar

REPOSITORIES: biostudies-other

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Genome-wide identification of targets for the archaeal heat shock regulator phr by cell-free transcription of genomic DNA.

Keese Annette M AM Schut Gerrit J GJ Ouhammouch Mohamed M Adams Michael W W MW Adams Michael W W MW Thomm Michael M

Journal of bacteriology 20091218 5

The hyperthermophilic archaeon Pyrococcus furiosus grows optimally near 100 degrees C and undergoes a heat shock response at 105 degrees C, mediated at least in part by the heat shock regulator Phr. Genes encoding a small heat shock protein (HSP20) and a member of the AAA(+) ATPase are the only known targets of the regulator, but a genetic mutant of Phr has yet to be characterized. We describe here an alternative approach for the identification of the regulon of Phr based on cell-free transcript ...[more]

PMID: 20023014

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Project description:BACKGROUND: Heat shock response in eukaryotes is transcriptionally regulated by conserved heat shock transcription factors (Hsfs). Hsf genes are represented by a large multigene family in plants and investigation of the Hsf gene family will serve to elucidate the mechanisms by which plants respond to stress. In recent years, reports of genome-wide structural and evolutionary analysis of the entire Hsf gene family have been generated in two model plant systems, Arabidopsis and rice. Maize, an important cereal crop, has represented a model plant for genetics and evolutionary research. Although some Hsf genes have been characterized in maize, analysis of the entire Hsf gene family were not completed following Maize (B73) Genome Sequencing Project. RESULTS: A genome-wide analysis was carried out in the present study to identify all Hsfs maize genes. Due to the availability of complete maize genome sequences, 25 nonredundant Hsf genes, named ZmHsfs were identified. Chromosomal location, protein domain and motif organization of ZmHsfs were analyzed in maize genome. The phylogenetic relationships, gene duplications and expression profiles of ZmHsf genes were also presented in this study. Twenty-five ZmHsfs were classified into three major classes (class A, B, and C) according to their structural characteristics and phylogenetic comparisons, and class A was further subdivided into 10 subclasses. Moreover, phylogenetic analysis indicated that the orthologs from the three species (maize, Arabidopsis and rice) were distributed in all three classes, it also revealed diverse Hsf gene family expression patterns in classes and subclasses. Chromosomal/segmental duplications played a key role in Hsf gene family expansion in maize by investigation of gene duplication events. Furthermore, the transcripts of 25 ZmHsf genes were detected in the leaves by heat shock using quantitative real-time PCR. The result demonstrated that ZmHsf genes exhibit different expression levels in heat stress treatment. CONCLUSIONS: Overall, data obtained from our investigation contributes to a better understanding of the complexity of the maize Hsf gene family and provides the first step towards directing future experimentation designed to perform systematic analysis of the functions of the Hsf gene family.

Project description:BACKGROUND:Heat shock transcription factor (Hsfs) is widely found in eukaryotes and prokaryotes. Hsfs can not only help organisms resist high temperature, but also participate in the regulation of plant growth and development (such as involved in the regulation of seed maturity and affects the root length of plants). The Hsf gene was first isolated from yeast and then gradually found in plants and sequenced, such as Arabidopsis thaliana, rice, maize. Tartary buckwheat is a rutin-rich crop, and its nutritional value and medicinal value are receiving more and more attention. However, there are few studies on the Hsf genes in Tartary buckwheat. With the whole genome sequence of Tartary buckwheat, we can effectively study the Hsf gene family in Tartary buckwheat. RESULTS:According to the study, 29 Hsf genes of Tartary buckwheat (FtHsf) were identified and renamed according to location of FtHsf genes on chromosome after removing a redundant gene. Therefore, only 29 FtHsf genes truly had the functional characteristics of the FtHsf family. The 29 FtHsf genes were located on 8 chromosomes of Tartary buckwheat, and we found gene duplication events in the FtHsf gene family, which may promote the expansion of the FtHsf gene family. Then, the motif compositions and the evolutionary relationship of FtHsf proteins and the gene structures, cis-acting elements in the promoter, synteny analysis of FtHsf genes were discussed in detail. What's more, we found that the transcription levels of FtHsf in different tissues and fruit development stages were significantly different by quantitative real-time PCR (qRT-PCR), implied that FtHsf may differ in function. CONCLUSIONS:In this study, only 29 Hsf genes were identified in Tartary buckwheat. Meanwhile, we also classified the FtHsf genes, and studied their structure, evolutionary relationship and the expression pattern. This series of studies has certain reference value for the study of the specific functional characteristics of Tartary buckwheat Hsf genes and to improve the yield and quality of Tartary buckwheat in the future.

Dataset Information

Genome-wide identification of targets for the archaeal heat shock regulator phr by cell-free transcription of genomic DNA.

Publications

Genome-wide identification of targets for the archaeal heat shock regulator phr by cell-free transcription of genomic DNA.

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