Abstract Background Bioinformatic genome surveys indicate that self-cleaving ribonucleic acids (ribozymes) appear to be widespread among all domains of life, although the functions of only a small number have been validated by biochemical methods. Alternatively, cell-based reporter gene assays can be used to validate ribozyme function. However, reporter activity can be confounded by phenomena unrelated to ribozyme-mediated cleavage of RNA. Results We established a ribozyme reporter system in Escherichia coli in which a significant reduction of reporter activity is manifest when an active ribozyme sequence is fused to the reporter gene and the expression of a foreign Bacillus subtilis RNaseJ1 5′ exonuclease is induced from a chromosomally-integrated gene in the same cell. Conclusions The reporter system could be useful for validating ribozyme function in candidate sequences identified from bioinformatics.
Background.Hypusination is an essential post-translational modification in eukaryotes.The two enzymes required for this modification, namely deoxyhypusine synthase (DHS) and deoxyhypusine hydrolase are also conserved.Plasmodium falciparum human malaria parasites possess genes for both hypusination enzymes, which are hypothesized to be targets of antimalarial drugs.Methods.Transgenic P. falciparum parasites with modification of the PF3D7_1412600 gene encoding PfDHS enzyme were created by insertion of the glmS riboswitch or the M9 inactive variant.The PfDHS protein was studied in transgenic parasites by confocal microscopy and Western immunoblotting.The biochemical function of PfDHS enzyme in parasites was assessed by hypusination and nascent protein synthesis assays.Gene essentiality was assessed by competitive growth assays and chemogenomic profiling.Results.Clonal transgenic parasites with integration of glmS riboswitch downstream of the PfDHS gene were established.PfDHS protein was present in the cytoplasm of transgenic parasites in asexual stages.The PfDHS protein could be attenuated five-fold in transgenic parasites with an active riboswitch, whereas PfDHS protein expression was unaffected in control transgenic parasites with insertion of the riboswitch-inactive sequence.Attenuation of PfDHS expression for 72 h led to a significant reduction of hypusinated protein; however, global protein synthesis was unaffected.Parasites with attenuated PfDHS expression showed a significant growth defect, although their decline was not as rapid as parasites with attenuated dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) expression.PfDHS-attenuated parasites showed increased sensitivity to N 1 -guanyl-1,7-diaminoheptane, a structural analog of spermidine, and a known inhibitor of DHS enzymes.Discussion.Loss of PfDHS function leads to reduced hypusination, which may be important for synthesis of some essential proteins.The growth defect in parasites with attenuated Pf DHS expression suggests that this gene is essential.However, the slower decline of PfDHS mutants compared with PfDHFR-TS mutants in competitive growth assays suggests that PfDHS is less vulnerable as an antimalarial target.Nevertheless, the data validate PfDHS as an antimalarial target which can be inhibited by spermidine-like compounds.
SUMMARY We have investigated the evolution of the bicoid ( bcd ) gene in fly species of the Muscoidea Superfamily. We obtained the complete bcd sequence from the housefly Musca domestica and found polymorphism in the coding region among Musca strains. In addition to Musca , we cloned most of the bcd coding sequences from two blowfly species Calliphora vicina and Lucilia sericata . The 5′ and 3′ regulatory regions flanking the Musca bcd gene are widely diverged in sequence from Drosophila; however, some important sequence motifs identified in Drosophila bcd are present. The predicted RNA secondary structures of the 3′ UTRs are similar, despite sequence divergence. Comparison of Bicoid (Bcd) proteins shows a serine‐rich domain of unknown function is present in the Muscoidea species, but is absent in other species. The in vivo function of bcd in Musca was tested by RNAi to mimic loss of function phenotype. We obtained a head defect phenotype similar to weak bcd alleles of Drosophila . Although our comparisons initially suggest functional conservation between species, closer inspection reveals significant differences. Divergence of structural motifs, such as regulatory elements in flanking regions and conservation of protein domains in some species but not in others, points to functional divergence between species. We suggest that the larger embryonic size in Muscoidea species restricts the morphogenetic activity of a weak Bcd activator, which has evolved a more specialized role in head determination and lost some functions in thoracic development.
RNA-seq data analysis from DHA treatment of P. berghei. Limma results from in vivo 2Â h treatments of mice infected P. berghei ANKA and injected with 10Â mg/kg DHA or vehicle control. (XLS 809Â kb)
Sugarcane is an important global food crop and energy resource. To facilitate the sugarcane improvement program, genome and gene information are important for studying traits at the molecular level. Most currently available transcriptome data for sugarcane were generated using second-generation sequencing platforms, which provide short reads. The de novo assembled transcripts from these data are limited in length, and hence may be incomplete and inaccurate, especially for long RNAs.We generated a transcriptome dataset of leaf tissue from a commercial Thai sugarcane cultivar Khon Kaen 3 (KK3) using PacBio RS II single-molecule long-read sequencing by the Iso-Seq method. Short-read RNA-Seq data were generated from the same RNA sample using the Ion Proton platform for reducing base calling errors.A total of 119,339 error-corrected transcripts were generated with the N50 length of 3,611 bp, which is on average longer than any previously reported sugarcane transcriptome dataset. 110,253 sequences (92.4%) contain an open reading frame (ORF) of at least 300 bp long with ORF N50 of 1,416 bp. The mean lengths of 5' and 3' untranslated regions in 73,795 sequences with complete ORFs are 1,249 and 1,187 bp, respectively. 4,774 transcripts are putatively novel full-length transcripts which do not match with a previous Iso-Seq study of sugarcane. We annotated the functions of 68,962 putative full-length transcripts with at least 90% coverage when compared with homologous protein coding sequences in other plants.The new catalog of transcripts will be useful for genome annotation, identification of splicing variants, SNP identification, and other research pertaining to the sugarcane improvement program. The putatively novel transcripts suggest unique features of KK3, although more data from different tissues and stages of development are needed to establish a reference transcriptome of this cultivar.
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