Molecular mechanisms that distinguish TFIID housekeeping from regulatable SAGA promoters

An important distinction is frequently made between constitutively expressed housekeeping genes versus regulated genes. Although generally characterized by different DNA elements, chromatin architecture and cofactors, it is not known to what degree promoter classes strictly follow regulatability rules and which molecular mechanisms dictate such differences. We show that SAGA‐dominated/TATA‐box promoters are more responsive to changes in the amount of activator, even compared to TFIID/TATA‐like promoters that depend on the same activator Hsf1. Regulatability is therefore an inherent property of promoter class. Further analyses show that SAGA/TATA‐box promoters are more dynamic because TATA‐binding protein recruitment through SAGA is susceptible to removal by Mot1. In addition, the nucleosome configuration upon activator depletion shifts on SAGA/TATA‐box promoters and seems less amenable to preinitiation complex formation. The results explain the fundamental difference between housekeeping and regulatable genes, revealing an additional facet of combinatorial control: an activator can elicit a different response dependent on core promoter class.     

Tomato and barley contain duplicated copies of cryptochrome 1

The cryptochrome family of blue‐light photoreceptors is involved in the control of plant photomorphogenesis and photoperiodic responses. Two cryptochromes have been described in Arabidopsis and tomato. To investigate the composition of the cryptochrome gene family in angiosperms, we used a ‘garden PCR’ approach, amplifying DNA from different plant species with the same pair of degenerated oligonucleotides representing conserved sequences from the flavin‐binding domain. Different numbers of Cry‐homologous sequences were found in different species: two each in Arabidopsis (Dicots, Brassicaceae), melon (Dicots, Cucurbitaceae) and banana tree (Monocots, Musaceae); three each in tomato (Dicots, Solanaceae) and barley (Monocots, Graminaceae). These sequences contain open reading frames (OFRs) with high homology to cryptochromes, but not photolyases, and are transcribed into RNA. In each case, a Cry1‐ and a Cry2‐like sequence was recognizable. The third gene of tomato and barley seems to have arisen from recent, independent duplications of Cry1, and was thus named Cry1b. The tomato Cry1b gene encodes a protein of 583 amino acids (the shortest of the three tomato cryptochromes), with a high similarity to Cry1. The C‐terminus of Cry1b is truncated before the conserved Ser‐Thr‐Ala‐Glu‐Ser‐Ser‐Ser (STAESSS) motif found in both Cry1a and Cry2. The Cry1b mRNA is expressed throughout the tomato plant, reaching maximal levels of expression in the flower (like Cry1a and Cry2). We conclude that tomato and barley contain at least one additional expressed member of the Cry1 gene family.     

Fractional Ca(2+) current through human neuronal alpha7 nicotinic acetylcholine receptors

The neuronal alpha7 nicotinic acetylcholine (ACh) receptor is believed to be a highly Ca(2+) permeable ligand-gated receptor-channel. However, the contribution of Ca(2+) to cationic current generated by ACh has not yet been directly measured to date. Simultaneous fluorescence and whole-cell current measurements using the Ca(2+) indicator dye fura-2 were made in GH4C1 pituitary cells stably expressing human alpha7 receptors and the fractional Ca(2+) current (the proportion of whole-cell current carried by Ca(2+); P(f)) was determined. We report that the P(f) value was 11.4+/-1.3%. This value was significantly larger than P(f) of human L248Talpha7 receptor mutant (P(f)=6.3+/-1.0%) and of rat alpha7 receptor (P(f)=8.8+/-1.5%) both determined in transiently transfected GH4C1 cells. In our knowledge, the findings here reported indicate the human alpha7 receptors are the most Ca(2+) conductive homomeric ligand-gated receptor-channels expressed in a heterologous cell system.     

The TP73 Gene Polymorphism (rs4648551, A>G) Is Associated with Diminished Ovarian Reserve

It’s known that the members of the TP53 family are involved in the regulation of female reproduction. Studies in mice showed that the TP73 gene (member of this family) plays a role in the size of follicular pool, ovulation rate and maintenance of genomic stability. In the present study we analyzed data from 605 patients with ≤ 37 years attending their first intracytoplasmic sperm injection (ICSI). The association between the TP73 polymorphism (rs4648551, A>G) and the following parameters related to ovarian reserve, like age, antral follicular count (AFC), anti-Mullerian hormone levels (AMH) and ovarian response prediction index (ORPI) was evaluated. Our results showed an association of the AA genotype with diminished ovarian reserve (AMH <1, AFC ≤9). Women presenting the AA genotype had a 2.0-fold increased risk for having AMH <1 and AFC ≤9 (OR 2.0, 95% CI 1.23-3.31, P = 0.005). Patients presenting AA genotype had the lowest levels of AMH (P = 0.02), the lowest number of antral follicles (P = 0.01) and the lowest ORPI (P = 0.007). Analyzing the alleles, we can see an enrichment of the A allele in the group of diminished ovarian reserve (OR 1.4, 95%CI 1.02-1.83, P = 0.04). To the best of our knowledge, the present study is the first to analyze this polymorphism in humans for assessing the numbers of ovarian follicles and AMH levels and, therefore, the ovarian reserve. Our findings can contribute to the use of this polymorphism as a potential marker of diminished ovarian reserve.     

Genome analysis of DNA repair genes in the alpha proteobacterium <Emphasis Type="Italic">Caulobacter crescentus</Emphasis>

Background  

The integrity of DNA molecules is fundamental for maintaining life. The DNA repair proteins protect organisms against genetic damage, by removal of DNA lesions or helping to tolerate them. DNA repair genes are best known from the gamma-proteobacterium Escherichia coli, which is the most understood bacterial model. However, genome sequencing raises questions regarding uniformity and ubiquity of these DNA repair genes and pathways, reinforcing the need for identifying genes and proteins, which may respond to DNA damage in other bacteria.     

A Nano-MgO and Ionic Liquid-Catalyzed ‘Green’ Synthesis Protocol for the Development of Adamantyl-Imidazolo-Thiadiazoles as Anti-Tuberculosis Agents Targeting Sterol 14α-Demethylase (CYP51)

In this work, we describe the ‘green’ synthesis of novel 6-(adamantan-1-yl)-2-substituted-imidazo[2,1-b][1,3,4]thiadiazoles (AITs) by ring formation reactions using 1-(adamantan-1-yl)-2-bromoethanone and 5-alkyl/aryl-2-amino1,3,4-thiadiazoles on a nano material base in ionic liquid media. Given the established activity of imidazothiadiazoles against M. tuberculosis, we next examined the anti-TB activity of AITs against the H₃₇Rv strain using Alamar blue assay. Among the tested compounds 6-(adamantan-1-yl)-2-(4-methoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazole (3f) showed potent inhibitory activity towards M. tuberculosis with an MIC value of 8.5 μM. The inhibitory effect of this molecule against M. tuberculosis was comparable to the standard drugs such as Pyrazinamide, Streptomycin, and Ciprofloxacin drugs. Mechanistically, an in silico analysis predicted sterol 14α-demethylase (CYP51) as the likely target and experimental activity of 3f in this system corroborated the in silico target prediction. In summary, we herein report the synthesis and biological evaluation of novel AITs against M. tuberculosis that likely target CYP51 to induce their antimycobacterial activity.     

The Rotary Zone Thermal Cycler: A Low-Power System Enabling Automated Rapid PCR

Advances in molecular biology, microfluidics, and laboratory automation continue to expand the accessibility and applicability of these methods beyond the confines of conventional, centralized laboratory facilities and into point of use roles in clinical, military, forensic, and field-deployed applications. As a result, there is a growing need to adapt the unit operations of molecular biology (e.g., aliquoting, centrifuging, mixing, and thermal cycling) to compact, portable, low-power, and automation-ready formats. Here we present one such adaptation, the rotary zone thermal cycler (RZTC), a novel wheel-based device capable of cycling up to four different fixed-temperature blocks into contact with a stationary 4-microliter capillary-bound sample to realize 1-3 second transitions with steady state heater power of less than 10 W. We demonstrate the utility of the RZTC for DNA amplification as part of a highly integrated rotary zone PCR (rzPCR) system that uses low-volume valves and syringe-based fluid handling to automate sample loading and unloading, thermal cycling, and between-run cleaning functionalities in a compact, modular form factor. In addition to characterizing the performance of the RZTC and the efficacy of different online cleaning protocols, we present preliminary results for rapid single-plex PCR, multiplex short tandem repeat (STR) amplification, and second strand cDNA synthesis.