The Function of a Spindle Checkpoint Gene bub-1 in C. elegans Development

BACKGROUND:The serine/threonine kinase BUB1 (Budding Uninhibited by Benzimidazole 1) was originally identified in yeast as a checkpoint protein, based on its mutant's incapacity of delaying the cell cycle in response to loss of microtubules. Our understanding of its function is primarily from studies carried out in yeast S. cerevisiae. It has been shown that it is a component of the mitotic spindle checkpoint and regulates the separation of sister chromatids through its downstream molecules. However, its roles in multi-cellular organisms remain unclear. METHODS AND FINDINGS:In nematode C. elegans, rapid cell divisions primarily occur in embryos and in germline of postembryonic larvae and adults. In addition, a select set of cells undergo a few rounds of cell division postembryonically. One common phenotype associated with impaired cell division is described as Stu (Sterile and Uncoordinated) [1], [2]. We conducted a genetic screen for zygotic mutants that displayed Stu phenotype in C. elegans. We isolated seven Stu mutants that fell into five complementation groups. We report here that two mutations, FanWang5 (fw5) and FanWang8 (fw8) affect the bub-1 gene, a homolog of yeast BUB1. Both mutant alleles of fw5 and fw8 exhibited variable behavioral defects, including developmental arrest, uncoordination and sterility. The number of postembryonically born neurons in the ventral cord decreased and their axon morphology was abnormal. Also, the decrease of neurons in the ventral cord phenotype could not be suppressed by a caspase-3 loss-of-function mutant. In addition, bub-1(fw5 and fw8) mutants showed widespread effects on postembryonic development in many cell lineages. We found that bub-1 functioned maternally in several developmental lineages at the embryonic stage in C. elegans. Studies in yeast have shown that BUB1 functions as a spindle checkpoint protein by regulating the anaphase promoting complex/cyclosome (APC/C). We performed double mutant analysis and observed that bub-1 genetically interacted with several downstream genes, including fzy-1/CDC20, mat-2/APC1 and emb-27/APC6. CONCLUSIONS:Our results demonstrate a conserved role of bub-1 in cell-cycle regulation and reveal that C. elegans bub-1 is required both maternally and zygotically. Further, our genetic analysis is consistent with that the function of bub-1 in C. elegans is likely similar to its yeast and mammalian homologs

Detection of intergenic non-coding RNAs expressed in the main developmental stages in Drosophila melanogaster

How many intergenically encoded non-coding RNAs (ncRNAs) are expressed during various developmental stages in Drosophila? Previous analyses in one or a few developmental stages indicated abundant expression of intergenic ncRNAs. However, some reported that ncRNAs have been recently falsified, and, in general, the false positive rate for ncRNA detection is unknown. In this report, we used reverse transcription-PCR (RT-PCR), a more robust method, to detect ncRNAs from the intergenic regions that are expressed in four major developmental stages (6–8 h embryo, 20–22 h embryo, larvae and adult). We tested 1027 regions, ∼10% of all intergenic regions, and detected transcription by RT–PCR. The results from 18 342 RT–PCR experiments revealed evidence for transcription in 72.7% of intergenic regions in the developmental process. The early developmental stage appears to be associated with more abundant ncRNAs than later developmental stages. In the early stage, we detected 43.6% of intergenic regions that encode transcripts in the triplicate RT–PCR experiments, yielding an estimate of 5006 intergenic regions in the entire genome likely encoding ncRNAs. We compared the RT–PCR-related approach with previous tiling array-based approach and observed that the latter method is insensitive to short ncRNAs, especially the molecules less than 120 bp. We measured false positive rates for the analyzed genomic approaches including the RT–PCR and tiling array method

Prediction of Cone Crusher Performance Considering Liner Wear

Cone crushers are used in the aggregates and mining industries to crush rock material. The pressure on cone crusher liners is the key factor that influences the hydraulic pressure, power draw and liner wear. In order to dynamically analyze and calculate cone crusher performance along with liner wear, a series of experiments are performed to obtain the crushed rock material samples from a crushing plant at different time intervals. In this study, piston die tests are carried out and a model relating compression coefficient, compression ratio and particle size distribution to a corresponding pressure is presented. On this basis, a new wear prediction model is proposed combining the empirical model for predicting liner wear with time parameter. A simple and practical model, based on the wear model and interparticle breakage, is presented for calculating compression ratio of each crushing zone along with liner wear. Furthermore, the size distribution of the product is calculated based on existing size reduction process model. A method of analysis of product size distribution and shape in the crushing process considering liner wear is proposed. Finally, the validity of the wear model is verified via testing. The result shows that there is a significant improvement of the prediction of cone crusher performance considering liner wear as compared to the previous model

Nuclease sensitive element binding protein 1 associates with the selenocysteine insertion sequence and functions in mammalian selenoprotein translation

Biosynthesis of selenium-containing proteins requires insertion of the unusual amino acid selenocysteine by alternative translation of a UGA codon, which ordinarily serves as a stop codon. In eukaryotes, selenoprotein translation depends upon one or more selenocysteine insertion sequence (SECIS) elements located in the 3\u27-untranslated region of the mRNA, as well as several SECIS-binding proteins. Our laboratory has previously identified nuclease sensitive element binding protein 1 (NSEP1) as another SECIS-binding protein, but evidence has been presented both for and against its role in SECIS binding in vivo and in selenoprotein translation. Our current studies sought to resolve this controversy, first by investigating whether NSEP1 interacts closely with SECIS elements within intact cells. After reversible in vivo cross-linking and ribonucleoprotein immunoprecipitation, mRNAs encoding two glutathione peroxidase family members co-precipitated with NSEP1 in both human and rat cell lines. Co-immunoprecipitation of an epitope-tagged GPX1 construct depended upon an intact SECIS element in its 3\u27-untranslated region. To test the functional importance of this interaction on selenoprotein translation, we used small inhibitory RNAs to reduce the NSEP1 content of tissue culture cells and then examined the effect of that reduction on the activity of a SECIS-dependent luciferase reporter gene for which expression depends upon readthrough of a UGA codon. Co-transfection of small inhibitory RNAs directed against NSEP1 decreased its expression by approximately 50% and significantly reduced luciferase activity. These studies demonstrate that NSEP1 is an authentic SECIS binding protein that is structurally associated with the selenoprotein translation complex and functionally involved in the translation of selenoproteins in mammalian cells

Nuclease sensitive element binding protein 1 gene disruption results in early embryonic lethality

Nuclease sensitive element binding protein 1 (NSEP1) is a member of the EFIA/NSEP1/YB-1 family of DNA-binding proteins whose members share a cold shock domain; it has also been termed DNA-binding protein B and Y box binding protein-1 because of its recognition of transcriptional regulatory elements. In addition, NSEP1 functions in the translational regulation of renin, ferritin, and interleukin 2 transcripts, and our laboratory has reported that it plays a role in the biosynthesis of selenium-containing proteins. To test the functional importance of NSEP1 in murine embryonic development, we have utilized a clone of ES cells in which the NSEP1 gene had been disrupted by integration of a plasmid gene-trapping vector into the seventh exon. Injection of these cells into C57BL/6 blastocysts resulted in 11 high percentage chimeric mice; crosses to wild type C57BL/6 mice generated 82 F1 agouti mice, indicating germ line transmission of the ES cell clone, but genotyping showed no evidence of the disrupted allele in any of these agouti offspring even though spermatozoa from four of five tested mice contained the targeted allele. Embryos harvested after timed matings of chimeric male mice demonstrated only the wildtype allele in 27 embryos tested at E7.5, E12.5, and E18.5. These results suggest that gene targeting of NSEP1 induces a lethal phenotype in early embryos, due to either haploinsufficiency of NSEP1 or formation of a dominant negative form of the protein. In either case, these data indicate the functional importance of the NSEP1 gene in murine early embryonic development

DNA Methylation and RNA-Sequencing Analysis to Identify Genes Related to Spontaneous Leaf Spots in a Wheat Variety ‘Zhongkenuomai No.1’

Greenish leaf variation has been reported widely as a trait of great interest in wheat for improving photosynthesis. Zhongkenuomai No.1 (ZKNM1), a mutant with recoverable leaf spots, was regarded as a suitable material for studying chlorophyll synthesis-related mechanisms. In this study, transcriptome and DNA methylation analyses were conducted in ZKNM1 leaves to determine the transcriptional regulatory mechanism of leaf spot development. Ultimately, 890 differentially expressed genes (DEGs) were discovered, with chlorophyll biosynthesis pathway genes downregulated and chlorophyll degradation pathway genes upregulated, possibly acting as a double block to chlorophyll accumulation. Among them, HEMA1s (Glutamyl-tRNA reductase family proteins) and PORAs (protochlorophyllide oxidoreductase A) were the most important controlled genes. Furthermore, a genome-wide methylation analysis indicates that a hypermethylated region is present 1690 bp upstream of the transcriptional start sites in spot tissues (SPs), and 131 DNA methylation-mediated DEGs were identified, one of which encoded a putative resistance gene (TraesCS1A02G009500) and was a hub gene in interaction network modules. In the sample groups with leaf spots (SPs), this gene may be involved in the photosynthetic processes. The findings indicated that dynamic variations in DNA methylation play key roles in gene regulation to govern leaf spot development