Mating type differentiation in tetrahymena thermophila: Characterization of the delayed refeeding effect and its implications concerning intranuclear coordination

Mating type determination in Tetrahymena thermophila involves developmentally programmed, heritable alterations of the macronucleus, localized to the mtd locus. This determination can be predictably controlled by the environmental conditions during macronuclear development, eg, temperature and time of refeeding. In this article we have further characterized the effects of delayed refeeding on mating type determination, as revealed by the frequency of mating types among the progeny of a cross. Our results show that 1) the magnitude of this starvation effect decreases with temperature of conjugation and becomes undetectable at 18°C; 2) starvation during the interval 14 to 22 hr (after conjugation is induced at 30°C) is a necessary and sufficient condition for the induction of starvation effects; 3) relative mating type frequencies vary monotonically with nutrient concentration present during this critical period; and 4) sister macronuclei, developing under starvation conditions in the same cytoplasm, differentiate majority mating types characteristic of early or late refeeding; sister macronuclei show no apparent correlation with each other. On the basis of our observations on early and late refed cells, we propose that the composition of the newly developed macronucleus is the outcome of two key events: 1) mating type determination at the mtd locus and 2) differential molecular cloning of generally one or two autonomously replicating fragments (ARFs) of the macronuclear DNA bearing the mtd locus.     

A set of Arabidopsis thaliana miRNAs involve shoot regeneration in vitro

Plant miRNAs, the critical regulator of gene expression, involve many development processes in vivo. However, the roles of miRNAs in plant cell proliferation and redifferntiation in vitro remain unknown. To determine better the molecular mechanism of these processes, we have recently reported that a set of miRNAs with different expression patterns between cells of totipotent and non-totipotent Arabidopsis calli. Some of these were specifically up- or downregulated during callus formation or shoot regeneration, and other development. Among them, miR160, and one of its target genes, ARF10, regulated Arabidopsis in vitro shoot regeneration via WUS, CLV3 and CUC1/2. The miR160-overexpressing, 35S transgenic lines, exhibited reduced shoot regeneration efficiency. The mARF10, a miR160-resistant form of ARF10, showed a high level of shoot regeneration ability. In the transgenic, expression of the above shoot meristem-specific genes was elevated, which is consistent with the improved shoot regeneration. In contrast, the ARF10 deficient knockout mutant produced fewer regenerated shoot. However, overexpressors of ARF10 were only marginally more efficient than the wild type with the respect to shoot regeneration. Our observation strongly supports that proper shoot regeneration from in vitro cultured cells requires the miR160-directed negative influence of ARF10. The enhanced expression of ARF10 is likely to have contributed to the improved regeneration ability.     

LncRNA TP73-AS1 is a novel regulator in cervical cancer via miR-329-3p/ARF1 axis

Cervical cancer holds one of the highest morbidity and mortality in various types of cancers. It even leads to the most number of cancer-related deaths of women. A lot of research has indicated that the anomalous expression of long noncoding RNAs (lncRNAs) would induce carcinogenesis and is associated with poor prognosis of patients with cancer. However, the function and mechanism of many lncRNAs still call for further research. Tumor Protein P73 Antisense RNA 1 (TP73-AS1) is no exception. LncRNA TP73-AS1 has been found to promote cancer progressions in various cancers. It is upregulated in cervical cancer cells. The proliferation and migration ability of cervical cancer cells can also be boosted by TP73-AS1 in return. Meanwhile, miRNA-329-3p is downregulated in cervical cancer cells and could bind with both TP73-AS1 and ADP Ribosylation Factor 1 (ARF1). TP73-AS1 inhibited miR-329-3p expression while miR-329-3p inhibited ARF1 expression. More importantly, TP73-AS1 can positively regulate ARF1 expression. Based on all these experiments, TP73-AS1 regulates ARF1 expression by competitively binding with miR-329-3p, thus regulating cervical cancer progression. Further rescue assays confirmed TP73-AS1 regulates cervical cell proliferation and migration via miR-329-3p/ARF1. TP73-AS1 might serve as a novel regulator in cervical cancer.     

The amphipathic helices of Arfrp1 and Arl14 are sufficient to determine subcellular localizations

The subcellular localization of Arf family proteins is generally thought to be determined by their corresponding guanine nucleotide exchange factors. By promoting GTP binding, guanine nucleotide exchange factors induce conformational changes of Arf proteins exposing their N-terminal amphipathic helices, which then insert into the membranes to stabilize the membrane association process. Here, we found that the N-terminal amphipathic motifs of the Golgi-localized Arf family protein, Arfrp1, and the endosome- and plasma membrane–localized Arf family protein, Arl14, play critical roles in spatial determination. Exchanging the amphipathic helix motifs between these two Arf proteins causes the switch of their localizations. Moreover, the amphipathic helices of Arfrp1 and Arl14 are sufficient for cytosolic proteins to be localized into a specific cellular compartment. The spatial determination mediated by the Arfrp1 helix requires its binding partner Sys1. In addition, the residues that are required for the acetylation of the Arfrp1 helix and the myristoylation of the Arl14 helix are important for the specific subcellular localization. Interestingly, Arfrp1 and Arl14 are recruited to their specific cellular compartments independent of GTP binding. Our results demonstrate that the amphipathic motifs of Arfrp1 and Arl14 are sufficient for determining specific subcellular localizations in a GTP-independent manner, suggesting that the membrane association and activation of some Arf proteins are uncoupled.     

Combined small RNA and degradome sequencing reveals microRNA regulation during immature maize embryo dedifferentiation

Genetic transformation of maize is highly dependent on the development of embryonic calli from the dedifferentiated immature embryo. To better understand the regulatory mechanism of immature embryo dedifferentiation, we generated four small RNA and degradome libraries from samples representing the major stages of dedifferentiation. More than 186 million raw reads of small RNA and degradome sequence data were generated. We detected 102 known miRNAs belonging to 23 miRNA families. In total, we identified 51, 70 and 63 differentially expressed miRNAs (DEMs) in the stage I, II, III samples, respectively, compared to the control. However, only 6 miRNAs were continually up-regulated by more than fivefold throughout the process of dedifferentiation. A total of 87 genes were identified as the targets of 21 DEM families. This group of targets was enriched in members of four significant pathways including plant hormone signal transduction, antigen processing and presentation, ECM-receptor interaction, and alpha-linolenic acid metabolism. The hormone signal transduction pathway appeared to be particularly significant, involving 21 of the targets. While the targets of the most significant DEMs have been proved to play essential roles in cell dedifferentiation. Our results provide important information regarding the regulatory networks that control immature embryo dedifferentiation in maize.     

Effect of cell cycle inhibitor p19ARF on senescence of human diploid cell

To investigate the effect of cell cycle inhibitor p19ARF on replicative senescence of human diploid cell, recombinant p19ARF eukaryotic expression vector was constructed and p19ARF gene was transfected into human diploid fibroblasts (WI-38 cells) by liposome-mediated transfection for overexpression. Then, the effects of p19ARF on replicative senescence of WI-38 cells were observed. The results re- vealed that, compared with control cells, the WI-38 cells in which p19ARF gene was introduced showed significant up-regulation of p53 and p21 expression level, decrease of cell generation by 10 12 generations, decline of cell growth rate with cell cycle being arrested at G1 phase, increase of positive rate of senescent marker SA-β-gal staining, and decrease of mitochondrial membrane potential. The morphology of the transfected fibroblasts presented the characteristics changes similar to senescent cells. These results indicated that high expression of p19ARF may promote the senescent process of human diploid cells.