A Genetic Analysis Of Folate Transport In Chinese Hamster Ovary Cells

Chinese Hamster Ovary cells that are about 50x more resistant to the cytotoxic action of methotrexate than wild-type cells were deficient in the ability to take up methotrexate. In the absence of any exogeneous folates these cells require 100-250x the level of folinic acid as do wild-type cells to support growth at a similar level. Two classes of mutants were distinguishable by their revertability for growth on folinic acid and ability to take-up folic acid. Revertants derived from one class were similar to wild-type cells in their ability to grow in medium containing low levels of folinic acid and in their sensitivity to methotrexate. In contrast, revertants from a second class were able to grow in medium containing low or no folinic acid, but retained their methotrexate resistance. Somatic cell hybrids formed between these two classes of mutants were non-complementing. These observations suggested that some but not all components may be shared between the transport systems mediating methotrexate and folic acid uptake.;The second class of methotrexate-resistant Chinese hamster ovary cells have been complemented to methotrexate sensitivity by transfection with DNA isolated from either wild-type Chinese hamster ovary or human G2 cells. Primary and secondary transfectants regained the ability to take up methotrexate in a manner similar to wild-type cells. To assist in cloning the sequences responsible for this complementation, transfections have also been carried out with DNA from a wild-type cosmid library. Transfectants have been isolated which have regained methotrexate sensitivity, the ability to take-up methotrexate and were found to contain a limited number of transfected cosmid sequences. Three cosmid clones have been isolated from a primary methotrexate sensitive transfectant cosmid library which after being transfected into the mutant, rescued the methotrexate resistant phenotype of the mutant at a high frequency. Restriction endonuclease analysis of the cosmid clones determined that they overlapped extensively and shared, two regions of 6.6 kB and 20.9 kB DNA. These observations suggested that a gene involved in Mtx uptake is contained within these regions. This is the first report of the molecular cloning of a gene specific to Mtx uptake

Active repression by unliganded retinoid receptors in development: less is sometimes more

The retinoid receptors have major roles throughout development, even in the absence of ligand. Here, we summarize an emerging theme whereby gene repression, mediated by unliganded retinoid receptors, can dictate cell fate. In addition to activating transcription, retinoid receptors actively repress gene transcription by recruiting cofactors that promote chromatin compaction. Two developmental processes for which gene silencing by the retinoid receptors is essential are head formation in Xenopus and skeletal development in the mouse. Inappropriate repression, by oncogenic retinoic acid (RA)* receptor (RAR) fusion proteins, blocks myeloid differentiation leading to a rare form of leukemia. Our current understanding of the developmental role of retinoid repression and future perspectives in this field are discussed

Requirement for RAR-mediated gene repression in skeletal progenitor differentiation

Chondrogenesis is a multistep process culminating in the establishment of a precisely patterned template for bone formation. Previously, we identified a loss in retinoid receptor–mediated signaling as being necessary and sufficient for expression of the chondroblast phenotype (Weston et al., 2000. J. Cell Biol. 148:679–690). Here we demonstrate a close association between retinoic acid receptor (RAR) activity and the transcriptional activity of Sox9, a transcription factor required for cartilage formation. Specifically, inhibition of RAR-mediated signaling in primary cultures of mouse limb mesenchyme results in increased Sox9 expression and activity. This induction is attenuated by the histone deacetylase inhibitor, trichostatin A, and by coexpression of a dominant negative nuclear receptor corepressor-1, indicating an unexpected requirement for RAR-mediated repression in skeletal progenitor differentiation

Genome-wide analyses of gene expression during mouse endochondral ossification

Background: Endochondral ossification is a complex process involving a series of events that are initiated by the establishment of a chondrogenic template and culminate in its replacement through the coordinated activity of osteoblasts, osteoclasts and endothelial cells. Comprehensive analyses of in vivo gene expression profiles during these processes are essential to obtain a complete understanding of the regulatory mechanisms involved. Methodology/Principal Findings: To address these issues, we completed a microarray screen of three zones derived from manually segmented embryonic mouse tibiae. Classification of genes differentially expressed between each respective zone, functional categorization as well as characterization of gene expression patterns, cytogenetic loci, signaling pathways and functional motifs both confirmed reported data and provided novel insights into endochondral ossification. Parallel comparisons of the microdissected tibiae data set with our previously completed micromass culture screen further corroborated the suitability of micromass cultures for modeling gene expression in chondrocyte development. The micromass culture system demonstrated striking similarities to the in vivo microdissected tibiae screen; however, the micromass system was unable to accurately distinguish gene expression differences in the hypertrophic and mineralized zones of the tibia. Conclusions/Significance: These studies allow us to better understand gene expression patterns in the growth plate and endochondral bones and provide an important technical resource for comparison of gene expression in diseased or experimentally-manipulated cartilages. Ultimately, this work will help to define the genomic context in which genes are expressed in long bones and to understand physiological and pathological ossification. © 2010 James et al

Genome-Wide Analyses of Gene Expression during Mouse Endochondral Ossification

Endochondral ossification is a complex process involving a series of events that are initiated by the establishment of a chondrogenic template and culminate in its replacement through the coordinated activity of osteoblasts, osteoclasts and endothelial cells. Comprehensive analyses of in vivo gene expression profiles during these processes are essential to obtain a complete understanding of the regulatory mechanisms involved.To address these issues, we completed a microarray screen of three zones derived from manually segmented embryonic mouse tibiae. Classification of genes differentially expressed between each respective zone, functional categorization as well as characterization of gene expression patterns, cytogenetic loci, signaling pathways and functional motifs both confirmed reported data and provided novel insights into endochondral ossification. Parallel comparisons of the microdissected tibiae data set with our previously completed micromass culture screen further corroborated the suitability of micromass cultures for modeling gene expression in chondrocyte development. The micromass culture system demonstrated striking similarities to the in vivo microdissected tibiae screen; however, the micromass system was unable to accurately distinguish gene expression differences in the hypertrophic and mineralized zones of the tibia.These studies allow us to better understand gene expression patterns in the growth plate and endochondral bones and provide an important technical resource for comparison of gene expression in diseased or experimentally-manipulated cartilages. Ultimately, this work will help to define the genomic context in which genes are expressed in long bones and to understand physiological and pathological ossification

Diffusion and spatial correlations in suspensions of swimming particles

Populations of swimming microorganisms produce fluid motions that lead to dramatically enhanced diffusion of tracer particles. Using simulations of suspensions of swimming particles in a periodic domain, we capture this effect and show that it depends qualitatively on the mode of swimming: swimmers ``pushed'' from behind by their flagella show greater enhancement than swimmers that are ``pulled'' from the front. The difference is manifested by an increase, that only occurs for pushers, of the diffusivity of passive tracers and the velocity correlation length with the size of the periodic domain. A physical argument supported by a mean field theory sheds light on the origin of these effects.Comment: 10 pages, 3 figures, to be published in Phys. Rev. Let

BMP action in skeletogenesis involves attenuation of retinoid signaling

The bone morphogenetic protein (BMP) and growth and differentiation factor (GDF) signaling pathways have well-established and essential roles within the developing skeleton in coordinating the formation of cartilaginous anlagen. However, the identification of bona fide targets that underlie the action of these signaling molecules in chondrogenesis has remained elusive. We have identified the gene for the retinoic acid (RA) synthesis enzyme Aldh1a2 as a principal target of BMP signaling; prochondrogenic BMPs or GDFs lead to attenuation of Aldh1a2 expression and, consequently, to reduced activation of the retinoid signaling pathway. Consistent with this, antagonism of retinoid signaling phenocopies BMP4 action, whereas RA inhibits the chondrogenic stimulatory activity of BMP4. BMP4 also down-regulates Aldh1a2 expression in organ culture and, consistent with this, Aldh1a2 is actively excluded from the developing cartilage anlagens. Collectively, these findings provide novel insights into BMP action and demonstrate that BMP signaling governs the fate of prechondrogenic mesenchyme, at least in part, through regulation of retinoid signaling

Massive migration from the steppe is a source for Indo-European languages in Europe

We generated genome-wide data from 69 Europeans who lived between 8,000-3,000 years ago by enriching ancient DNA libraries for a target set of almost four hundred thousand polymorphisms. Enrichment of these positions decreases the sequencing required for genome-wide ancient DNA analysis by a median of around 250-fold, allowing us to study an order of magnitude more individuals than previous studies and to obtain new insights about the past. We show that the populations of western and far eastern Europe followed opposite trajectories between 8,000-5,000 years ago. At the beginning of the Neolithic period in Europe, ~8,000-7,000 years ago, closely related groups of early farmers appeared in Germany, Hungary, and Spain, different from indigenous hunter-gatherers, whereas Russia was inhabited by a distinctive population of hunter-gatherers with high affinity to a ~24,000 year old Siberian6 . By ~6,000-5,000 years ago, a resurgence of hunter-gatherer ancestry had occurred throughout much of Europe, but in Russia, the Yamnaya steppe herders of this time were descended not only from the preceding eastern European hunter-gatherers, but from a population of Near Eastern ancestry. Western and Eastern Europe came into contact ~4,500 years ago, as the Late Neolithic Corded Ware people from Germany traced ~3/4 of their ancestry to the Yamnaya, documenting a massive migration into the heartland of Europe from its eastern periphery. This steppe ancestry persisted in all sampled central Europeans until at least ~3,000 years ago, and is ubiquitous in present-day Europeans. These results provide support for the theory of a steppe origin of at least some of the Indo-European languages of Europe