Analysis of candidate imprinted genes linked to Dlk1-Gtl2 using a congenic mouse line

The study of genomic imprinting requires the use of DNA sequence polymorphisms between interfertile mouse species or strains. Most commonly, crosses between Mus musculus domesticus and Mus musculus castaneus or Mus spretus animals are used. Difficulties arise in the maintenance of these wild-derived mice in conventional animal facilities, however, and can be overcome by the use of a congenic strain for the region under study. We describe here the generation of a new mouse line, congenic for a region on distal Chromosome (Chr) 12 that encompasses the Dlk1–Gtl2 imprinted domain. We have taken a first step towards demonstrating the utility of these animals by assaying known genes located within the congenic interval for imprinted expression. We show that the two genes located immediately proximal to Dlk1, the Yy1 and Wars genes, are expressed in a biallelic manner. In addition, we have analyzed the Dio3 gene, located distal to Gtl2. This gene displays preferential expression of the paternal allele, with approximately 75% of the total message level originating from the paternal allele and 25% originating from the maternal allele. These data delineate the position of the Wars gene as the proximal boundary of the Dlk1–Gtl2 imprinted domain, and identify Dio3 as another potentially imprinted gene within this domain.     

Cdc5 influences phosphorylation of Net1 and disassembly of the RENT complex

Background  

In S. cerevisiae, the mitotic exit network (MEN) proteins, including the Polo-like protein kinase Cdc5 and the protein phosphatase Cdc14, are required for exit from mitosis. In pre-anaphase cells, Cdc14 is sequestered to the nucleolus by Net1 as a part of the RENT complex. When cells are primed to exit mitosis, the RENT complex is disassembled and Cdc14 is released from the nucleolus.     

Tumor cell growth inhibition and extracellular signal-regulated kinase (ERK) phosphorylation by novel K vitamins.

2-(2-hydroxy-ethylsulfanyl)-3-methyl-1,4-naphthoquinone or CPD-5, a K vitamin analog, was previously indicated to be a potent growth inhibitor for Hep 3B hepatoma cells in vitro. Here, we show that CPD-5 and two newly synthesized analogs, 2-(2-hydroxy-ethylsulfanyl)-3-methyl-5- nitro-1,4-naphthoquinone (PD-37) and 2-(2-hydroxy-ethylsulfanyl)-3- methyl-5-acetylamino-1,4-naphthoquinone (PD-42), are potent growth inhibitors of 13 different human cancer cell lines, with IC50 values in the range of 3-54 microM. Phospho-ERK was induced by each of three K vitamin analogs in every cell line in a dose-dependent manner, at growth inhibitory doses. ERK phosphorylation and growth inhibitory effects were strongly correlated, with p=0.0080 for CPD-5, p=0.0076 for PD-37 and p=0.0251 for PD-42. The induction of phospho-ERK and growth inhibition were antagonized by thiol-containing anti-oxidants, but not by catalase, consistent with a possible arylating mechanism. The data show a novel class of growth inhibitors with a wide spectrum of action that induces ERK hyper-phosphorylation, as a possible new growth inhibitory feature.     

Detection of QTL controlling digestive efficiency and anatomy of the digestive tract in chicken fed a wheat-based diet

Background

Improving digestive efficiency is a major goal in poultry production, to reduce production costs, make possible the use of alternative feedstuffs and decrease the volume of manure produced. Since measuring digestive efficiency is difficult, identifying molecular markers associated with genes controlling this trait would be a valuable tool for selection. Detection of QTL (quantitative trait loci) was undertaken on 820 meat-type chickens in a F2 cross between D- and D+ lines divergently selected on low or high AMEn (apparent metabolizable energy value of diet corrected to 0 nitrogen balance) measured at three weeks in animals fed a low-quality diet. Birds were measured for 13 traits characterizing digestive efficiency (AMEn, coefficients of digestive utilization of starch, lipids, proteins and dry matter (CDUS, CDUL, CDUP, CDUDM)), anatomy of the digestive tract (relative weights of the proventriculus, gizzard and intestine and proventriculus plus gizzard (RPW, RGW, RIW, RPGW), relative length and density of the intestine (RIL, ID), ratio of proventriculus and gizzard to intestine weight (PG/I); and body weight at 23 days of age. Animals were genotyped for 6000 SNPs (single nucleotide polymorphisms) distributed on 28 autosomes, the Z chromosome and one unassigned linkage group.

Results

Nine QTL for digestive efficiency traits, 11 QTL for anatomy-related traits and two QTL for body weight at 23 days of age were detected. On chromosome 20, two significant QTL at the genome level co-localized for CDUS and CDUDM, i.e. two traits that are highly correlated genetically. Moreover, on chromosome 16, chromosome-wide QTL for AMEn, CDUS, CDUDM and CDUP, on chromosomes 23 and 26, chromosome-wide QTL for CDUS, on chromosomes 16 and 26, co-localized QTL for digestive efficiency and the ratio of intestine length to body weight and on chromosome 27 a chromosome-wide QTL for CDUDM were identified.

Conclusions

This study identified several regions of the chicken genome involved in the control of digestive efficiency. Further studies are necessary to identify the underlying genes and to validate these in commercial populations and breeding environments.     

Mitochondrial impairment observed in fibroblasts from South African Parkinson’s disease patients with parkin mutations

Parkinson’s disease (PD), defined as a neurodegenerative disorder, is characterized by the loss of dopaminergic neurons in the substantia nigra in the midbrain. Loss-of-function mutations in the parkin gene are a major cause of autosomal recessive, early-onset PD. Parkin has been implicated in the maintenance of healthy mitochondria, although previous studies show conflicting findings regarding mitochondrial abnormalities in fibroblasts from patients harboring parkin-null mutations. The aim of the present study was to determine whether South African PD patients with parkin mutations exhibit evidence for mitochondrial dysfunction. Fibroblasts were cultured from skin biopsies obtained from three patients with homozygous parkin-null mutations, two heterozygous mutation carriers and two wild-type controls. Muscle biopsies were obtained from two of the patients. The muscle fibers showed subtle abnormalities such as slightly swollen mitochondria in focal areas of the fibers and some folding of the sarcolemma. Although no differences in the degree of mitochondrial network branching were found in the fibroblasts, ultrastructural abnormalities were observed including the presence of electron-dense vacuoles. Moreover, decreased ATP levels which are consistent with mitochondrial dysfunction were observed in the patients’ fibroblasts compared to controls. Remarkably, these defects did not manifest in one patient, which may be due to possible compensatory mechanisms. These results suggest that parkin-null patients exhibit features of mitochondrial dysfunction. Involvement of mitochondria as a key role player in PD pathogenesis will have important implications for the design of new and more effective therapies.     

Broadening horizons: holistic viewpoints from the Biology of Genomes

A report on the Cold Spring Harbor Laboratory 27th annual meeting on the Biology of Genomes, held in Cold Spring Harbor, New York, USA, 6-10 May 2014.     

Wintering birds avoid warm sunshine: predation and the costs of foraging in sunlight

Wintering birds can gain significant thermal benefits by foraging in direct sunlight. However, exposure to bright sunlight might make birds easier to detect by predators and may also cause visual glare that can reduce a bird’s ability to monitor the environment. Thus, birds likely experience a trade-off between the thermal benefits and predation-related costs of foraging in direct sunlight. To examine this possible thermoregulation-predation trade-off, we monitored the behavior of mixed-species flocks of wintering emberizid sparrows foraging in alternating strips of sunlight and shade. On average, these sparrows routinely preferred to forage in the shade, despite midday air temperatures as much as 30 °C below their thermoneutral zone. This preference for shade was strongest at relatively high temperatures when the thermal benefits of foraging in sunlight were reduced, suggesting a thermoregulation-predation trade-off. Glare could be reduced if birds faced away from the sun while feeding in direct sunlight, but we found that foraging birds tended to face southward (the direction of the sun). We speculate that other factors, such as the likely direction of predator approach, may explain this southerly orientation, particularly if predators use solar glare to their advantage during an attack. This interpretation is supported by the fact that birds had the weakest southerly orientation on cloudy days. Wintering birds may generally avoid foraging in direct sunlight to minimize their risk of predation. However, given the thermal benefits of sunshine, such birds may benefit from foraging in habitats that provide a mosaic of sunlit and shaded microhabitats.     

Development and Characterization of Pepducins as Gs-biased Allosteric Agonists

The β₂-adrenergic receptor (β₂AR) is a prototypical G protein-coupled receptor that mediates many hormonal responses, including cardiovascular and pulmonary function. β-Agonists used to combat hypercontractility in airway smooth muscle stimulate β₂AR-dependent cAMP production that ultimately promotes airway relaxation. Chronic stimulation of the β₂AR by long acting β-agonists used in the treatment of asthma can promote attenuated responsiveness to agonists and an increased frequency of fatal asthmatic attacks. β₂AR desensitization to β-agonists is primarily mediated by G protein-coupled receptor kinases and β-arrestins that attenuate receptor-G_s coupling and promote β₂AR internalization and degradation. A biased agonist that can selectively stimulate G_s signaling without promoting receptor interaction with G protein-coupled receptor kinases and β-arrestins should serve as an advantageous asthma therapeutic. To identify such molecules, we screened ∼50 lipidated peptides derived from the intracellular loops of the β₂AR, known as pepducins. This screen revealed two classes of G_s-biased pepducins, receptor-independent and receptor-dependent, as well as several β-arrestin-biased pepducins. The receptor-independent G_s-biased pepducins operate by directly stimulating G protein activation. In contrast, receptor-dependent G_s-biased pepducins appear to stabilize a G_s-biased conformation of the β₂AR that couples to G_s but does not undergo G protein-coupled receptor kinase-mediated phosphorylation or β-arrestin-mediated internalization. Functional studies in primary human airway smooth muscle cells demonstrate that G_s-biased pepducins are not subject to conventional desensitization and thus may be good candidates for the development of next generation asthma therapeutics. Our study reports the first G_s-biased activator of the β₂AR and provides valuable tools for the study of β₂AR function.     

Coordinate regulation of RARgamma2, TBP, and TAFII135 by targeted proteolysis during retinoic acid-induced differentiation of F9 embryonal carcinoma cells

Background  

Treatment of mouse F9 embryonal carcinoma cells with all-trans retinoic acid (T-RA) induces differentiation into primitive endodermal type cells. Differentiation requires the action of the receptors for all trans, and 9cis-retinoic acid (RAR and RXR, respectively) and is accompanied by growth inhibition, changes in cell morphology, increased apoptosis, proteolytic degradation of the RARγ2 receptor, and induction of target genes.