Switchgrass (Panicum virgatum L.) polyubiquitin gene (PvUbi1 and PvUbi2) promoters for use in plant transformation

<p>Abstract</p> <p>Background</p> <p>The ubiquitin protein is present in all eukaryotic cells and promoters from ubiquitin genes are good candidates to regulate the constitutive expression of transgenes in plants. Therefore, two switchgrass (<it>Panicum virgatum </it>L.) ubiquitin genes (<it>PvUbi1 </it>and <it>PvUbi2</it>) were cloned and characterized. Reporter constructs were produced containing the isolated 5' upstream regulatory regions of the coding sequences (i.e. <it>PvUbi1 </it>and <it>PvUbi2 </it>promoters) fused to the <it>uidA </it>coding region (<it>GUS</it>) and tested for transient and stable expression in a variety of plant species and tissues.</p> <p>Results</p> <p><it>PvUbi1 </it>consists of 607 bp containing <it>cis</it>-acting regulatory elements, a 5' untranslated region (UTR) containing a 93 bp non-coding exon and a 1291 bp intron, and a 918 bp open reading frame (ORF) that encodes four tandem, head -to-tail ubiquitin monomer repeats followed by a 191 bp 3' UTR. <it>PvUbi2 </it>consists of 692 bp containing <it>cis</it>-acting regulatory elements, a 5' UTR containing a 97 bp non-coding exon and a 1072 bp intron, a 1146 bp ORF that encodes five tandem ubiquitin monomer repeats and a 183 bp 3' UTR. <it>PvUbi1 </it>and <it>PvUbi2 </it>were expressed in all examined switchgrass tissues as measured by qRT-PCR. Using biolistic bombardment, <it>PvUbi1 </it>and <it>PvUbi2 </it>promoters showed strong expression in switchgrass and rice callus, equaling or surpassing the expression levels of the CaMV <it>35S, 2x35S, ZmUbi1</it>, and <it>OsAct1 </it>promoters. GUS staining following stable transformation in rice demonstrated that the <it>PvUbi1 </it>and <it>PvUbi2 </it>promoters drove expression in all examined tissues. When stably transformed into tobacco (<it>Nicotiana tabacum</it>), the <it>PvUbi2+3 </it>and <it>PvUbi2+9 </it>promoter fusion variants showed expression in vascular and reproductive tissues.</p> <p>Conclusions</p> <p>The <it>PvUbi1 </it>and <it>PvUbi2 </it>promoters drive expression in switchgrass, rice and tobacco and are strong constitutive promoter candidates that will be useful in genetic transformation of monocots and dicots.</p

Comparative Omics-Driven Genome Annotation Refinement: Application across Yersiniae

Genome sequencing continues to be a rapidly evolving technology, yet most downstream aspects of genome annotation pipelines remain relatively stable or are even being abandoned. The annotation process is now performed almost exclusively in an automated fashion to balance the large number of sequences generated. One possible way of reducing errors inherent to automated computational annotations is to apply data from omics measurements (i.e. transcriptional and proteomic) to the un-annotated genome with a proteogenomic-based approach. Here, the concept of annotation refinement has been extended to include a comparative assessment of genomes across closely related species. Transcriptomic and proteomic data derived from highly similar pathogenic Yersiniae (Y. pestis CO92, Y. pestis Pestoides F, and Y. pseudotuberculosis PB1/+) was used to demonstrate a comprehensive comparative omic-based annotation methodology. Peptide and oligo measurements experimentally validated the expression of nearly 40% of each strain's predicted proteome and revealed the identification of 28 novel and 68 incorrect (i.e., observed frameshifts, extended start sites, and translated pseudogenes) protein-coding sequences within the three current genome annotations. Gene loss is presumed to play a major role in Y. pestis acquiring its niche as a virulent pathogen, thus the discovery of many translated pseudogenes, including the insertion-ablated argD, underscores a need for functional analyses to investigate hypotheses related to divergence. Refinements included the discovery of a seemingly essential ribosomal protein, several virulence-associated factors, a transcriptional regulator, and many hypothetical proteins that were missed during annotation

Long non-coding RNAs: spatial amplifiers that control nuclear structure and gene expression

Over the past decade, it has become clear that mammalian genomes encode thousands of long non-coding RNAs (lncRNAs), many of which are now implicated in diverse biological processes. Recent work studying the molecular mechanisms of several key examples — including Xist, which orchestrates X chromosome inactivation — has provided new insights into how lncRNAs can control cellular functions by acting in the nucleus. Here we discuss emerging mechanistic insights into how lncRNAs can regulate gene expression by coordinating regulatory proteins, localizing to target loci and shaping three-dimensional (3D) nuclear organization. We explore these principles to highlight biological challenges in gene regulation, in which lncRNAs are well-suited to perform roles that cannot be carried out by DNA elements or protein regulators alone, such as acting as spatial amplifiers of regulatory signals in the nucleus