Modulation of the agonist activity of antisteroids by a novel cis-acting element.

The amount of agonist activity displayed by the antiglucocorticoid dexamethasone mesylate (Dex-Mes) for the induction of tyrosine aminotransferase (TAT) in rat hepatoma cells is greater than for glutamine synthetase and varies over a period of weeks. This variation, which has been reproduced over a period of 40 h by changing the density of the cells, suggests the involvement of a trans-acting factor. The target of this proposed trans-acting factor has now been localized to the region between -3.9 to -2.9 of the rat TAT gene from experiments with cells that were stably transfected with hybrid TAT/CAT constructs. Deletion experiments with transiently transfected TAT/tk promoter/CAT constructs revealed that this entire activity could be conveyed by a 21 bp sequence of the TAT gene. Gel shift experiments support the binding of a factor(s) to this 21 bp sequence. Thus the activity of the antagonist Dex-Mes is relatively independent of steroid structure and is largely determined by the further interactions of a trans-acting factor with the cis-acting sequence. We call this novel sequence a glucocorticoid modulatory element. A model is advanced which accounts for almost all of the results concerning TAT induction by glucocorticoids. This same model may also be useful in explaining why the amount of agonist activity of most antisteroids varies, even for different genes within the same cell.     

MLV integration site selection is driven by strong enhancers and active promoters

Retroviruses integrate into the host genome in patterns specific to each virus. Understanding the causes of these patterns can provide insight into viral integration mechanisms, pathology and genome evolution, and is critical to the development of safe gene therapy vectors. We generated murine leukemia virus integrations in human HepG2 and K562 cells and subjected them to second-generation sequencing, using a DNA barcoding technique that allowed us to quantify independent integration events. We characterized >3 700 000 unique integration events in two ENCODE-characterized cell lines. We find that integrations were most highly enriched in a subset of strong enhancers and active promoters. In both cell types, approximately half the integrations were found in <2% of the genome, demonstrating genomic influences even narrower than previously believed. The integration pattern of murine leukemia virus appears to be largely driven by regions that have high enrichment for multiple marks of active chromatin; the combination of histone marks present was sufficient to explain why some strong enhancers were more prone to integration than others. The approach we used is applicable to analyzing the integration pattern of any exogenous element and could be a valuable preclinical screen to evaluate the safety of gene therapy vectors.     

Identification of a cis-acting element of human dihydrofolate reductase mRNA

Human dihydrofolate reductase (DHFR) is a critical target in cancer chemotherapy. Previous studies showed that an 82-nt RNA fragment within the DHFR mRNA protein-coding region functions as a DHFR cis-acting response element. In this study, we further investigated the key elements contained within this sequence that are required for the DHFR mRNA-DHFR protein interaction. Using enzymatic foot-printing assays and RNA-binding experiments, we isolated a 27-nt sequence (DHFR27, corresponding to nts 407-433), which bound with high affinity and specificity to human DHFR to form a ribonucleoprotein complex. In vivo transient transfection experiments using a luciferase reporter system revealed that DHFR27 RNA could repress the luciferase expression in a DHFR-dependent manner when placed upstream of luciferase mRNA. This work provides new insights into the essential molecular elements that mediate RNA-protein interactions.     

Adenylate cyclase regulates elongation of mammalian primary cilia

The primary cilium is a non-motile microtubule-based structure that shares many similarities with the structures of flagella and motile cilia. It is well known that the length of flagella is under stringent control, but it is not known whether this is true for primary cilia. In this study, we found that the length of primary cilia in fibroblast-like synoviocytes, either in log phase culture or in quiescent state, was confined within a range. However, when lithium was added to the culture to a final concentration of 100 mM, primary cilia of synoviocytes grew beyond this range, elongating to a length that was on average approximately 3 times the length of untreated cilia. Lithium is a drug approved for treating bipolar disorder. We dissected the molecular targets of this drug, and observed that inhibition of adenylate cyclase III (ACIII) by specific inhibitors mimicked the effects of lithium on primary cilium elongation. Inhibition of GSK-3β by four different inhibitors did not induce primary cilia elongation. ACIII was found in primary cilia of a variety of cell types, and lithium treatment of these cell types led to their cilium elongation. Further, we demonstrate that different cell types displayed distinct sensitivities to the lithium treatment. However, in all cases examined primary cilia elongated as a result of lithium treatment. In particular, two neuronal cell types, rat PC-12 adrenal medulla cells and human astrocytes, developed long primary cilia when lithium was used at or close to the therapeutic relevant concentration (1–2 mM). These results suggest that the length of primary cilia is controlled, at least in part, by the ACIII–cAMP signaling pathway.     

Regulation of peptide-chain elongation in mammalian cells.

The elongation phase of mRNA translation is the stage at which the polypeptide is assembled and requires a substantial amount of metabolic energy. Translation elongation in mammals requires a set of nonribosomal proteins called eukaryotic elongation actors or eEFs. Several of these proteins are subject to phosphorylation in mammalian cells, including the factors eEF1A and eEF1B that are involved in recruitment of amino acyl-tRNAs to the ribosome. eEF2, which mediates ribosomal translocation, is also phosphorylated and this inhibits its activity. The kinase acting on eEF2 is an unusual and specific one, whose activity is dependent on calcium ions and calmodulin. Recent work has shown that the activity of eEF2 kinase is regulated by MAP kinase signalling and by the nutrient-sensitive mTOR signalling pathway, which serve to activate eEF2 in response to mitogenic or hormonal stimuli. Conversely, eEF2 is inactivated by phosphorylation in response to stimuli that increase energy demand or reduce its supply. This likely serves to slow down protein synthesis and thus conserve energy under such circumstances.     

Activity of two strong promoters cloned into Bacillus subtilis

Two DNA fragments, one encoding the Escherichia coli trc promoter and the other encoding a sequence from the early region of Bacillus subtilis phage SPO1, were cloned into the B. subtilis promoter-probe vector pPL603. Both fragments effected strong in vivo promoter activity in vegetative B. subtilis cells.