5S rRNA genes from Saccharomyces cerevisiae were examined by Miller chromatin spreading, representing the first quantitative analysis of RNA polymerase III genes in situ by electron microscopy. These very short genes, ϳ132 nucleotides (nt), were engaged by one to three RNA polymerases. Analysis in different growth conditions and in strains with a fourfold range in gene copy number revealed regulation at two levels: number of active genes and polymerase loading per gene. Repressive growth conditions (presence of rapamycin or postexponential growth) led first to fewer active genes, followed by lower polymerase loading per active gene. The polymerase III elongation rate was estimated to be in the range of 60 to 75 nt/s, with a reinitiation interval of ϳ1.2 s. The yeast La protein, Lhp1, was associated with 5S genes. Its absence had no discernible effect on the amount or size of 5S RNA produced yet resulted in more polymerases per gene on average, consistent with a non-rate-limiting role for Lhp1 in a process such as polymerase release/recycling upon transcription termination.In eukaryotes, nuclear RNA transcription is carried out by one of three specialized RNA polymerases, Pol I, II, and III, with Pol III dedicated to making short, untranslated RNAs, including tRNAs, 5S rRNA, and many others (12). Consistent with the central role of these RNAs in protein synthesis, Pol III is an essential enzyme whose activity is regulated by various growth control pathways and is deregulated in human cancer cells and during cardiac hypertrophy (23, 67). Many years of study have led to detailed knowledge of the different types of promoters and of the trans-acting factors involved in Pol III gene expression (20,31,56). For the Saccharomyces cerevisiae 5S RNA genes studied herein, three factors are required: TFIIIA, which specifically binds the internal promoter of 5S genes, and TFIIIB and C, which are required for all yeast Pol III genes. Preinitiation complex formation begins with the deposition of TFIIIA on the internal promoter, followed by recruitment of the six-subunit TFIIIC complex, which contacts TFIIIA as well as sequences within the gene (Fig. 1A). TFIIIC recruits the three-subunit TFIIIB complex, which binds a conserved upstream regulatory sequence and is necessary and sufficient for Pol III transcription (30). TFIIIB, which contains TATA binding protein, recruits Pol III to the promoter and plays an essential role in the formation of the open initiation complex. The stability of TFIIIB, which restructures the DNA and recruits Pol III through multiple rounds of transcription (7,30), renders these genes very highly transcribed and the most prominent binding sites for TATA binding protein in the cell (51). The internally bound factors do not form a barrier to Pol III elongation.We used Miller chromatin spreading (39) to directly visualize the 5S gene population in actively growing yeast cells. This electron microscopy (EM) method has frequently been applied to the study of Pol I and Pol II gene expression. Pol III genes, h...

Background Root parasitic weeds are a major constraint to crop production worldwide causing significant yearly losses in yield and economic value. These parasites cause their destruction by attaching to their hosts with a unique organ, the haustorium, that allows them to obtain the nutrients (sugars, amino acids, etc.) needed to complete their lifecycle. Parasitic weeds differ in their nutritional requirements and degree of host dependency and the differential expression of sugar transporters is likely to be a critical component in the parasite’s post-attachment survival. Results We identified gene families encoding monosaccharide transporters (MSTs), sucrose transporters (SUTs), and SWEETs (Sugars Will Eventually be Exported Transporters) in three root-parasitic weeds differing in host dependency: Triphysaria versicolor (facultative hemiparasite), Phelipanche aegyptiaca (holoparasite), and Striga hermonthica (obligate hemiparasite). The phylogenetic relationship and differential expression profiles of these genes throughout parasite development were examined to uncover differences existing among parasites with different levels of host dependence. Differences in estimated gene numbers are found among the three parasites, and orthologs within the different sugar transporter gene families are found to be either conserved among the parasites in their expression profiles throughout development, or to display parasite-specific differences in developmentally-timed expression. For example, MST genes in the pGLT clade express most highly before host connection in Striga and Triphysaria but not Phelipanche , whereas genes in the MST ERD6-like clade are highly expressed in the post-connection growth stages of Phelipanche but highest in the germination and reproduction stages in Striga. Whether such differences reflect changes resulting from differential host dependence levels is not known. Conclusions While it is tempting to speculate that differences in estimated gene numbers and expression profiles among members of MST, SUT and SWEET gene families in Phelipanche , Striga and Triphysaria reflect the parasites’ levels of host dependence, additional evidence that altered transporter gene expression is causative versus consequential is needed. Our findings identify potential targets for directed manipulation that will allow for a better understanding of the nutrient transport process and perhaps a means for controlling the devastating effects of these parasites on crop productivity. Electronic supplementary material The online version of this article (10.1186/s12870-019-1786-y) contains supplementary material, which is available t...

Cancer immunotherapy is ineffective in low TMB EGFRmutant lung adenocarcinoma. Qi et al. performed a comprehensive proteogenomic profiling of HLA class I-presented immunopeptides in high TMB melanoma and low TMB EGFRmutant lung cancer. Similar numbers of immunopeptides were identified from both. Variant, CG antigen, PTM, and lncRNAderived peptides were identified. A novel strategy to identify lncRNA-derived peptides was developed. The direct identification of class I-presented immunopeptides will potentially accelerate precision immunotherapy for low TMB tumors. Highlights• Proteogenomics identified~35,000 class I-presented peptides.• CG antigen and PTM peptides identified in melanoma and lung cancer.• De novo search identified variant and lncRNA-derived peptides.• A new strategy to identify class I-presented lncRNA-derived peptides developed.

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BackgroundCowpea (Vigna unguiculata (L.) Walp.) is the most important food and forage legume in the semi-arid tropics of sub-Saharan Africa where approximately 80% of worldwide production takes place primarily on low-input, subsistence farm sites. Among the major goals of cowpea breeding and improvement programs are the rapid manipulation of agronomic traits for seed size and quality and improved resistance to abiotic and biotic stresses to enhance productivity. Knowing the suite of transcription factors (TFs) and transcriptionally active proteins (TAPs) that control various critical plant cellular processes would contribute tremendously to these improvement aims.ResultsWe used a computational approach that employed three different predictive pipelines to data mine the cowpea genome and identified over 4400 genes representing 136 different TF and TAP families. We compare the information content of cowpea to two evolutionarily close species common bean (Phaseolus vulgaris), and soybean (Glycine max) to gauge the relative informational content. Our data indicate that correcting for genome size cowpea has fewer TF and TAP genes than common bean (4408 / 5291) and soybean (4408/ 11,065). Members of the GROWTH-REGULATING FACTOR (GRF) and Auxin/indole-3-acetic acid (Aux/IAA) gene families appear to be over-represented in the genome relative to common bean and soybean, whereas members of the MADS (Minichromosome maintenance deficient 1 (MCM1), AGAMOUS, DEFICIENS, and serum response factor (SRF)) and C2C2-YABBY appear to be under-represented. Analysis of the AP2-EREBP APETALA2-Ethylene Responsive Element Binding Protein (AP2-EREBP), NAC (NAM (no apical meristem), ATAF1, 2 (Arabidopsis transcription activation factor), CUC (cup-shaped cotyledon)), and WRKY families, known to be important in defense signaling, revealed changes and phylogenetic rearrangements relative to common bean and soybean that suggest these groups may have evolved different functions.ConclusionsThe availability of detailed information on the coding capacity of the cowpea genome and in particular the various TF and TAP gene families will facilitate future comparative analysis and development of strategies for controlling growth, differentiation, and abiotic and biotic stress resistances of cowpea.Electronic supplementary materialThe online version of this article (10.1186/s12864-017-4306-1) contains supplementary material, which is available to authorized users.