The Swi5 activator recruits the Mediator complex to the HO promoter without RNA polymerase II

Regulation of HO gene expression in the yeast Saccharomyces cerevisiae is intricately orchestrated by an assortment of gene-specific DNA-binding and non-DNA binding regulators. Binding of the early G(1) transcription factor Swi5 to the distal URS1 element of the HO promoter initiates a cascade of events through recruitment of the Swi/Snf and SAGA complexes. In late G(1), binding of transcription factor SBF to promoter proximal sequences results in the timely expression of HO. In this work we describe an important additional layer of complexity to the current model by identifying a connection between Swi5 and the Mediator/RNA polymerase II holoenzyme complex. We show that Swi5 recruits Mediator to HO by specific interaction with the Gal11 module of the Mediator complex. Importantly, binding of both the Gal11 and Srb4 mediator components to the upstream region of HO is independent of the SBF factor. Swi/Snf is required for Mediator binding, and genetic suppression experiments suggest that Swi/Snf and Mediator act in the same genetic pathway of HO activation. Experiments examining the kinetics of binding show that Mediator binds to HO promoter elements 1.5 kb upstream of the transcription start site in early G(1), but this binding occurs without RNA Pol II. RNA Pol II does not bind to HO until late G(1), when HO is actively transcribed, and binding occurs exclusively to the TATA region

H-ras-1 restriction fragment length polymorphism in normal individuals and oral cancer patients in India

Restriction fragment length polymorphism (RFLP) of the human H-ras-l gene has been indicated as a marker for detection of individuals at high risk of cancer. We have investigated the association of RFLP at the H-ras-l locus and susceptibility to oral cancer by Southern hybridization analysis in 77 primary oral tumors and 99 healthy donors. The frequency distribution of the Bum HI fragments of H-ras-l revealed homozygous or heterozygous alleles in the two sub-populations. The heterozygous genotype occurred more frequently in the normal subjects (53%) as compared to the cancer patients (36%). Four common alleles Cl to C4, were noted predominantly in both groups, with rare alleles detected at a lower frequency. The common allele with 7.6 kb BamHI fragment was significantly higher in normals (10%) than in the tumor population (4%) (P≤0.05). However, a similar distribution of rare alleles in both groups indicated that the presence of rare alleles is not indicative of predisposition to oral cancer

Detection and cloning of potent transforming gene(s) from chewing tobacco-related human oral carcinomas

High molecular weight DNA isolated from 14 primary tumour tissues of human oral carcinoma patients was analysed for transforming activity by NIH3T3 co-transfection assay using pSV2neo gene as a selectable marker, followed by nude mouse tumorigenicity assay. Ten of the patient tumour tissues demonstrated molecular lesions in myc, ras or/and EGF-R genes, whereas 4 patients did not show tumour associated aberrations in these oncogenes. The G418-resistant transfected cells from 12 of 14 individual patients demonstrated transforming potential by colony formation in soft agar and tumour induction in nude mice within 25-80 days. DNAs from the transfected cells, consequent nude mice tumours and corresponding cell lines, contained human Alu sequences. Southern blot hybridisation with ras, myc, EGF-R oncogenes demonstrated the presence of human H-ras oncogene in one of the 12 sets of nude mice tumours. In contrast, DNA from the other 11 sets of nude mice tumours indicated absence of c-myc, N-myc, L-myc, H-ras, K-ras, N-ras and EGF-R genes on Southern analysis. Further, DNAs from five first cycle tumorigenic transformants were subjected to a second cycle of transfection, and induced tumours in nude mice with a shorter latency period of 21-50 days. The secondary transformants contained discrete human Alu sequences; however, the DNA did not hybridise with myc/ras/EGF-R probes. A genomic library was constructed from a second cycle nude mice tumour, using EMBL-3 as the vector. Four human Alu sequence positive clones were isolated on screening 2 × 105 plaques, and one of the recombinant clones subjected to fine restriction mapping using 16 restriction enzymes. The lack of association of the nude mice tumour DNA with myc/ras/EGF-R showing aberrations in the primary human tumour, implies activation of an alternative potent transforming gene(s) in the chewing tobacco-related oral carcinomas in India

Small molecule suppressors of Drosophila kinesin deficiency rescue motor axon development in a zebrafish model of spinal muscular atrophy.

Proximal spinal muscular atrophy (SMA) is the most common inherited motor neuropathy and the leading hereditary cause of infant mortality. Currently there is no effective treatment for the disease, reflecting a need for pharmacologic interventions that restore performance of dysfunctional motor neurons or suppress the consequences of their dysfunction. In a series of assays relevant to motor neuron biology, we explored the activities of a collection of tetrahydroindoles that were reported to alter the metabolism of amyloid precursor protein (APP). In Drosophila larvae the compounds suppressed aberrant larval locomotion due to mutations in the Khc and Klc genes, which respectively encode the heavy and light chains of kinesin-1. A representative compound of this class also suppressed the appearance of axonal swellings (alternatively termed axonal spheroids or neuritic beads) in the segmental nerves of the kinesin-deficient Drosophila larvae. Given the importance of kinesin-dependent transport for extension and maintenance of axons and their growth cones, three members of the class were tested for neurotrophic effects on isolated rat spinal motor neurons. Each compound stimulated neurite outgrowth. In addition, consistent with SMA being an axonopathy of motor neurons, the three axonotrophic compounds rescued motor axon development in a zebrafish model of SMA. The results introduce a collection of small molecules as pharmacologic suppressors of SMA-associated phenotypes and nominate specific members of the collection for development as candidate SMA therapeutics. More generally, the results reinforce the perception of SMA as an axonopathy and suggest novel approaches to treating the disease

Rescue of motor axon development by axonotrophic compounds; combined results.

<p>(A) The results with 2 µM SBL-154 were averaged across the experiments in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074325#pone-0074325-g007" target="_blank">Figure 7A–F</a> for both the control (vehicle-treated embryos) and experimental conditions, and mean values are plotted along with SEM (N = 6). SBL-154 significantly reduced the severity of motor axon defects (p<0.0001, N = 6; Mann-Whitney U test). (B) Suppression of Smn knockdown by SBL-185 was tested in 5 experiments, each of which examined 3 conditions: 0.5 µM SBL-185 (95 embryos scored among the 5 experiments, with about 20 embryos per experiment), 1 µM SBL-185 (95 embryos), and DMSO vehicle (99 embryos). Both concentrations of SBL-185 significantly reduce the severity of motor neuron dysmorphism (p<0.0001, N = 5; Kruskal-Wallis one-way ANOVA). (C) Suppression of Smn knockdown by SBL-190 was tested in 5 experiments, each of which examined 3 conditions: 1 µM SBL-190 (88 embryos), 2 µM SBL-190 (88 embryos), and DMSO vehicle (99 embryos). Both concentrations of SBL-190 significantly reduce the severity of motor neuron dysmorphism (p<0.0001, N = 5; Kruskal-Wallis one-way ANOVA). (D) Suppression of motor axon defects in <i>topped^b458</i> mutants by SBL-154 was tested in 3 experiments in which mutants were treated with either DMSO (60 embryos) or 2 µM SBL-154 (60 embryos). SBL-154 showed no effect (p = 0.14; Mann-Whitney U test).</p

Stimulation of neurite outgrowth in rat spinal motor neurons.

<p>Primary cultures of rat embryonic spinal motor neurons were treated with BDNF at 3.7 nM and with SBL-154, SBL-185, and SBL-190 at the two indicated concentrations of each compound. One-way ANOVA with Dunnett’s post test indicates that the positive control BDNF and each compound significantly enhanced neurite outgrowth relative to DMSO vehicle (N = 3 cultures; • p<0.001; <b>○</b> p<0.01; * p<0.05).</p