Uncovering New Functions for MicroRNAs in \u3cem\u3eCaenorhabditis elegans\u3c/em\u3e

In the race to understand microRNA (miRNA) functions in development and physiology, Caenorhabditis elegans investigators were the first out of the gate with the cloning and analysis of the lin-4 and let-7 miRNAs [1,2]. The starting point of strong, penetrant loss of function phenotypes facilitated these advancements. However, subsequent functional analysis of miRNAs in C. elegans was hampered by the lack of easily observable loss-of-function phenotypes [3]. There are several possible models to account for this observation. First, redundancy between related miRNAs can account for the absence of phenotypes in mutants missing individual miRNA genes [4,5]. Second, miRNAs may also function redundantly with unrelated miRNAs or other regulatory mechanisms. Third, identification of miRNA functions may require the analysis of specific cells during development, assays typically not included in initial broad phenotypic analyses. For example, the lsy-6 miRNA is an essential regulator of a chemosensory neuron cell fate in C. elegans [6]. Such a specialized function would not have been identified in broad phenotypic analyses. Finally, miRNAs may act to ‘fine-tune’ gene expression, to maintain protein levels of targets in an optimal range. Loss of this relatively minor regulatory input by miRNAs would not be expected to result in penetrant, observable defects under normal conditions. Recent work has analyzed the functions of individual miRNAs under conditions of environmental or physiological stress. With these approaches, functions for individual miRNAs, which remain elusive under normal growth conditions, have been uncovered. These stresses can be introduced through genetic mutations, environmental perturbations, or through the normal aging process. These results are consistent with the hypothesis that miRNAs act to ensure the robustness of developmental or physiological pathways [7]

Age-related cellular copper dynamics in the fungal ageing model Podospora anserina and in ageing human fibroblasts

In previous investigations an impact of cellular copper homeostasis on ageing of the ascomycete Podospora anserina has been demonstrated. Here we provide new data indicating that mitochondria play a major role in this process. Determination of copper in the cytosolic fraction using total reflection X-ray fluorescence spectroscopy analysis and eGfp reporter gene studies indicate an age-related increase of cytosolic copper levels. We show that components of the mitochondrial matrix (i.e. eGFP targeted to mitochondria) become released from the organelle during ageing. Decreasing the accessibility of mitochondrial copper in P. anserina via targeting a copper metallothionein to the mitochondrial matrix was found to result in a switch from a copper-dependent cytochrome-c oxidase to a copper-independent alternative oxidase type of respiration and results in lifespan extension. In addition, we demonstrate that increased copper concentrations in the culture medium lead to the appearance of senescence biomarkers in human diploid fibroblasts (HDFs). Significantly, expression of copper-regulated genes is induced during in vitro ageing in medium devoid of excess copper suggesting that cytosolic copper levels also increase during senescence of HDFs. These data suggest that the identified molecular pathway of age-dependent copper dynamics may not be restricted to P. anserina but may be conserved from lower eukaryotes to humans

Cloning of the Neurodegeneration Gene \u3cem\u3eDrop-Dead\u3c/em\u3e and Characterization of Additional Phenotypes of its Mutation

Mutations in the Drosophila gene drop-dead (drd) result in early adult lethality and neurodegeneration, but the molecular identity of the drd gene and its mechanism of action are not known. This paper describes the characterization of a new X-linked recessive adult-lethal mutation, originally called lot\u27s wife (lwf1) but subsequently identified as an allele of drd (drdlwf); drdlwf mutants die within two weeks of eclosion. Through mapping and complementation, the drd gene has been identified as CG33968, which encodes a putative integral membrane protein of unknown function. The drdlwf allele is associated with a nonsense mutation that eliminates nearly 80% of the CG33968 gene product; mutations in the same gene were also found in two previously described drd alleles. Characterization of drdlwf flies revealed additional phenotypes of drd, most notably, defects in food processing by the digestive system and in oogenesis. Mutant flies store significantly more food in their crops and defecate less than wild-type flies, suggesting that normal transfer of ingested food from the crop into the midgut is dependent upon the DRD gene product. The defect in oogenesis results in the sterility of homozygous mutant females and is associated with a reduction in the number of vitellogenic egg chambers. The disruption in vitellogenesis is far more severe than that seen in starved flies and so is unlikely to be a secondary consequence of the digestive phenotype. This study demonstrates that mutation of the drd gene CG33968 results in a complex phenotype affecting multiple physiological systems within the fly

Consequences of Postnatally Elevated Insulin-Like Growth Factor-II in Transgenic Mice: Endocrine Changes and Effects on Body and Organ Growth.

Insulin-like growth factor-II (IGF-II) is an important regulator of embryonic growth and differentiation, but its function in postnatal life is unclear. To address this point, we generated transgenic mice harboring fusion genes in which a human IGF-II complementary DNA is placed under the transcriptional control of the rat phosphoenolpyruvate carboxykinase promoter. Transgene-specific messenger RNA was detected in liver, kidney, and several parts of the gut. Serum IGF-II levels in transgenic mice were 2-3 times higher than those in controls and increased after starvation. Circulating IGF-I correlated negatively and IGF-binding protein-2 (IGFBP-2) positively with IGF-II levels, suggesting that IGF-I is displaced from IGFBPs by IGF-II and that IGFII is a major regulator of IGFBP-2. Serum levels of IGFBP-3 and IGFBP-4 tended to be higher in phosphoenolpyruvate carboxykinase- IGF-II transgenic mice than in controls, as evaluated by ligand blot analysis. Starvation reduced serum IGF-I, but increased IGFBP-2 in transgenic mice more markedly than in controls. Fasting insulin levels were significantly reduced in transgenic mice, whereas glucose levels were not influenced by elevated IGF-II. The body growth of 4- and 12- week-old mice was not significantly influenced by elevated IGF-II, but transgenic mice displayed increased kidney and testis weight at the age of 4 weeks, and increased adrenal weight at the age of 12 weeks. Our results demonstrate that elevated IGF-II in postnatal life has multiple endocrine consequences and subtle time-specific effects on organ growth

Molecular cytogenetic differentiation of paralogs of Hox paralogs in duplicated and re-diploidized genome of the North American paddlefish (Polyodon spathula).

BackgroundAcipenseriformes is a basal lineage of ray-finned fishes and comprise 27 extant species of sturgeons and paddlefishes. They are characterized by several specific genomic features as broad ploidy variation, high chromosome numbers, presence of numerous microchromosomes and propensity to interspecific hybridization. The presumed palaeotetraploidy of the American paddlefish was recently validated by molecular phylogeny and Hox genes analyses. A whole genome duplication in the paddlefish lineage was estimated at approximately 42 Mya and was found to be independent from several genome duplications evidenced in its sister lineage, i.e. sturgeons. We tested the ploidy status of available chromosomal markers after the expected rediploidization. Further we tested, whether paralogs of Hox gene clusters originated from this paddlefish specific genome duplication are cytogenetically distinguishable.ResultsWe found that both paralogs HoxA alpha and beta were distinguishable without any overlapping of the hybridization signal - each on one pair of large metacentric chromosomes. Of the HoxD, only the beta paralog was unequivocally identified, whereas the alpha paralog did not work and yielded only an inconclusive diffuse signal. Chromosomal markers on three diverse ploidy levels reflecting different stages of rediploidization were identified: quadruplets retaining their ancestral tetraploid condition, semi-quadruplets still reflecting the ancestral tetraploidy with clear signs of advanced rediploidization, doublets were diploidized with ancestral tetraploidy already blurred. Also some of the available microsatellite data exhibited diploid allelic band patterns at their loci whereas another locus showed more than two alleles.ConclusionsOur exhaustive staining of paddlefish chromosomes combined with cytogenetic mapping of ribosomal genes and Hox paralogs and with microsatellite data, brings a closer look at results of the process of rediploidization in the course of paddlefish genome evolution. We show a partial rediploidization represented by a complex mosaic structure comparable with segmental paleotetraploidy revealed in sturgeons (Acipenseridae). Sturgeons and paddlefishes with their high propensity for whole genome duplication thus offer suitable animal model systems to further explore evolutionary processes that were shaping the early evolution of all vertebrates

High-Throughput Microarray Detection of Vomeronasal Receptor Gene Expression in Rodents

We performed comprehensive data mining to explore the vomeronasal receptor (V1R and V2R) repertoires in mouse and rat using the mm5 and rn3 genome, respectively. This bioinformatic analysis was followed by investigation of gene expression using a custom designed high-density oligonucleotide array containing all of these receptors and other selected genes of interest. This array enabled us to detect the specific expression of V1R and V2Rs which were previously identified solely based on computational prediction from gene sequence data, thereby establishing that these genes are indeed part of the vomeronasal system, especially the V2Rs. One hundred sixty-eight V1Rs and 98 V2Rs were detected to be highly enriched in mouse vomeronasal organ (VNO), and 108 V1Rs and 87 V2Rs in rat VNO. We monitored the expression profile of mouse VR genes in other non-VNO tissues with the result that some VR genes were re-designated as VR-like genes based on their non-olfactory expression pattern. Temporal expression profiles for mouse VR genes were characterized and their patterns were classified, revealing the developmental dynamics of these so-called pheromone receptors. We found numerous patterns of temporal expression which indicate possible behavior-related functions. The uneven composition of VR genes in certain patterns suggests a functional differentiation between the two types of VR genes. We found the coherence between VR genes and transcription factors in terms of their temporal expression patterns. In situ hybridization experiments were performed to evaluate the cell number change over time for selected receptor genes

Progressive obesity leads to altered ovarian gene expression in the Lethal Yellow mouse: a microarray study

<p>Abstract</p> <p>Background</p> <p>Lethal yellow (LY; C57BL/6J <it>A</it>^<it>y</it>/<it>a</it>) mice exhibit adult-onset obesity, altered metabolic regulation, and early reproductive senescence. The present study was designed to test the hypothesis that obese LY mice possess differences in expression of ovarian genes relative to age-matched lean mice.</p> <p>Methods</p> <p>90- and 180-day-old LY and lean black (C57BL/6J <it>a/a</it>) mice were suppressed with GnRH antagonist (Antide^®), then stimulated with 5 IU eCG. cRNA derived from RNA extracts of whole ovarian homogenates collected 36 h post-eCG were run individually on Codelink Mouse Whole Genome Bioarrays (GE Healthcare Life Sciences).</p> <p>Results</p> <p>Fifty-two genes showed ≥ 2-fold differential (p < 0.05) expression between 180-day-old obese LY and lean black mice. LY mice exhibited elevated ovarian expression of agouti (350×), leptin (6.5×), and numerous genes involved in cholesterol/lipid transport and metabolism, e.g. lanosterol synthase, <it>Cyp51</it>, and steroidogenic acute regulatory protein (<it>Star</it>). Fewer genes showed lower expression in LY mice, e.g. angiotensinogen. In contrast, none of these genes showed differential expression in 90-day-old LY and black mice, which are of similar body weight. Interestingly, 180-day-old LY mice had a 2-fold greater expression of 11beta-hydroxysteroid dehydrogenase type 1 (<it>Hsd11b1</it>) and a 2-fold lesser expression of 11beta-hydroxysteroid dehydrogenase type 2 (<it>Hsd11b2</it>), differences not seen in 90-day-old mice. Consistent with altered <it>Hsd11b </it>gene expression, ovarian concentrations of corticosterone (C) were elevated in aging LY mice relative to black mice, but C levels were similar in young LY and black mice.</p> <p>Conclusion</p> <p>The data suggest that reproductive dysfunction in aging obese mice is related to modified intraovarian gene expression that is directly related to acquired obesity.</p