Browsing by Subject "expression"

Now showing 1 - 6 of 6
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    Bifurcation Analysis of Gene Regulatory Circuits Subject to Copy Number Variation
    (2010) Mileyko, Yuriy; Weitz, Joshua S
    Gene regulatory networks are comprised of many small gene circuits. Understanding expression dynamics of gene circuits for broad ranges of parameter space may provide insight into the behavior of larger regulatory networks as well as facilitate the use of circuits as autonomous units performing specific regulatory tasks. In this paper, we consider three common gene circuits and investigate the dependence of gene expression dynamics on the circuit copy number. In particular, we perform a detailed bifurcation analysis of the circuits' corresponding nonlinear gene regulatory models restricted to protein-only dynamics. Employing a geometric approach to bifurcation theory, we are able to derive closed form expressions for conditions which guarantee existence of saddle-node bifurcations caused by variation in the circuit copy number or copy number concentration. This result shows that the drastic effect of copy number variation on equilibrium behavior of gene circuits is highly robust to variation in other parameters in the circuits. We discuss a possibility of extending the current results to higher dimensional models which incorporate more details of the gene regulatory process.
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    Interchangeable Domains in the Kdo Transferases of Escherichia coli and Haemophilus influenzae
    (2010) Chung, Hak Suk; Raetz, Christian RH
    Kdo(2)-lipid A, a conserved substructure of lipopolysaccharide, plays critical roles in Gram-negative bacterial survival and interaction with host organisms. Inhibition of Kdo biosynthesis in Escherichia coli results in cell death and accumulation of the tetra-acylated precursor lipid IVA. E. coil KdtA (EcKdtA) is a bifunctional enzyme that transfers two Kdo units from two CMP-Kdo molecules to lipid IVA. In contrast, Haemophilia influenzae KdtA (HiKdtA) transfers only one Kdo unit. E. coil CMR300, which lacks Kdo transferase because of a deletion in kdtA, can be rescued to grow in broth at 37 degrees C if multiple copies of msbA are provided in trans. MsbA, the inner membrane transporter for nascent lipopolysaccharide, prefers hexa-acylated to tetra-acylated lipid A, but with the excess MsbA present in CMR300, lipid IVA is efficiently exported to the outer membrane. CMR300 is hypersensitive to hydrophobic antibiotics and bile salts and does not grow at 42 degrees C. Expressing HiKdtA in CMR300 results in the accumulation of Kdo-lipid IVA in place of lipid IVA without suppression of its growth phenotypes at 30 degrees C. EcKdtA restores intact lipopolysaccharide, together with normal antibiotic resistance, detergent resistance, and growth at 42 degrees C. To determine which residues are important for the mono- or bifunctional character of KdtA, protein chimeras were constructed using EcKdtA and HiKdtA. These chimeras, which are catalytically active, were characterized by in vitro assays and in vivo complementation. The N-terminal half of KdtA, especially the first 30 amino acid residues, specifies whether one or two Kdo units are transferred to lipid IVA.
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    Molecular characterization of numr-1 and numr-2: genes that increase both resistance to metal-induced stress and lifespan in Caenorhabditis elegans
    (2010) Tvermoes, Brooke E; Boyd, Windy A; Freedman, Jonathan H
    To define the mechanisms involved in the molecular response to the carcinogenic metal cadmium, two novel metal-inducible genes from C. elegans were characterized: numr-1 and numr-2 (nuclear localized metal responsive). numr-1 and numr-2 sequences and cellular patterns of expression are identical, indicating that these are functionally equivalent genes. Constitutive transcription of numr-1 and numr-2 is developmentally regulated and occurs in the intestine, in head and tail neurons, and vulva muscles. Exposure to metals induces numr-1 and numr-2 transcription in pharyngeal and intestinal cells. Other environmental stressors do not affect transcription, indicating that these are metal-specific, stress-responsive genes. NUMR-1 and NUMR-2 target to nuclei and colocalize with HSF-1, suggesting that they may be components of nuclear stress granules. Nematodes overexpressing NUMR-1 and NUMR-2 are resistant to stress and live longer than control animals; likewise reducing expression increases sensitivity to metals and decreases neuromuscular functions. Upstream regulatory regions of both genes contain potential binding sites for DAF-16 and SKN-1, which are components of the insulin-IGF-like signaling pathway. This pathway regulates longevity and stress responses in C. elegans. NUMR-1 and NUMR-2 may function to promote resistance to environmental stressors and longevity, which is mediated by the insulin-IGF-like signaling pathway.
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    Precise pattern of recombination in serotonergic and hypothalamic neurons in a Pdx1-cre transgenic mouse line
    (2010) Honig, Gerard; Liou, Angela; Berger, Miles; German, Michael S; Tecott, Laurence H
    Background: Multicellular organisms are characterized by a remarkable diversity of morphologically distinct and functionally specialized cell types. Transgenic techniques for the manipulation of gene expression in specific cellular populations are highly useful for elucidating the development and function of these cellular populations. Given notable similarities in developmental gene expression between pancreatic beta cells and serotonergic neurons, we examined the pattern of Cre-mediated recombination in the nervous system of a widely used mouse line, Pdx1-cre (formal designation, Tg(Ipf1-cre)89.1Dam), in which the expression of Cre recombinase is driven by regulatory elements upstream of the pdx1 (pancreatic-duodenal homeobox 1) gene. Methods: Single (hemizygous) transgenic mice of the pdx1-cre(Cre/0) genotype were bred to single (hemizygous) transgenic reporter mice (Z/EG and rosa26R lines). Recombination pattern was examined in offspring using whole-mount and sectioned histological preparations at e9.5, e10.5, e11.5, e16.5 and adult developmental stages. Results: In addition to the previously reported pancreatic recombination, recombination in the developing nervous system and inner ear formation was observed. In the central nervous system, we observed a highly specific pattern of recombination in neuronal progenitors in the ventral brainstem and diencephalon. In the rostral brainstem (r1-r2), recombination occurred in newborn serotonergic neurons. In the caudal brainstem, recombination occurred in non-serotonergic cells. In the adult, this resulted in reporter expression in the vast majority of forebrain-projecting serotonergic neurons (located in the dorsal and median raphe nuclei) but in none of the spinal cord-projecting serotonergic neurons of the caudal raphe nuclei. In the adult caudal brainstem, reporter expression was widespread in the inferior olive nucleus. In the adult hypothalamus, recombination was observed in the arcuate nucleus and dorsomedial hypothalamus. Recombination was not observed in any other region of the central nervous system. Neuronal expression of endogenous pdx1 was not observed. Conclusions: The Pdx1-cre mouse line, and the regulatory elements contained in the corresponding transgene, could be a valuable tool for targeted genetic manipulation of developing forebrain-projecting serotonergic neurons and several other unique neuronal sub-populations. These results suggest that investigators employing this mouse line for studies of pancreatic function should consider the possible contributions of central nervous system effects towards resulting phenotypes.
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    Regulation of male germ cell cycle arrest and differentiation by DND1 is modulated by genetic background
    (2011) Cook, Matthew S; Munger, Steven C; Nadeau, Joseph H; Capel, Blanche
    Human germ cell tumors show a strong sensitivity to genetic background similar to Dnd1(Ter/Ter) mutant mice, where testicular teratomas arise only on the 129/SvJ genetic background. The introduction of the Bax mutation onto mixed background Dnd1(Ter/Ter) mutants, where teratomas do not typically develop, resulted in a high incidence of teratomas. However, when Dnd1(Ter/Ter); Bax(-/-) double mutants were backcrossed to C57BL/6J, no tumors arose. Dnd1(Ter/Ter) germ cells show a strong downregulation of male differentiation genes including Nanos2. In susceptible strains, where teratomas initiate around E15.5-E17.5, many mutant germ cells fail to enter mitotic arrest in G0 and do not downregulate the pluripotency markers NANOG, SOX2 and OCT4. We show that DND1 directly binds a group of transcripts that encode negative regulators of the cell cycle, including p27(Kip1) and p21(Cip1). P27(Kip1) and P21(Cip1) protein are both significantly decreased in Dnd1(Ter/Ter) germ cells on all strain backgrounds tested, strongly suggesting that DND1 regulates mitotic arrest in male germ cells through translational regulation of cell cycle genes. Nonetheless, in C57BL/6J mutants, germ cells arrest prior to M-phase of the cell cycle and downregulate NANOG, SOX2 and OCT4. Consistent with their ability to rescue cell cycle arrest, C57BL/6J germ cells overexpress negative regulators of the cell cycle relative to 129/SvJ. This work suggests that reprogramming of pluripotency in germ cells and prevention of tumor formation requires cell cycle arrest, and that differences in the balance of cell cycle regulators between 129/SvJ and C57BL/6 might underlie differences in tumor susceptibility.
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    Uncoupling skeletal and connective tissue patterning: conditional deletion in cartilage progenitors reveals cell-autonomous requirements for Lmx1b in dorsal-ventral limb patterning
    (2010) Li, Ying; Qiu, Qiong; Watson, Spenser S; Schweitzer, Ronen; Johnson, Randy L
    Integration of muscle, connective tissue and skeletal patterning during development is essential for proper functioning of the musculoskeletal system. How this integration is achieved is poorly understood. There is ample evidence suggesting that skeletal pattern is programmed autonomously, whereas muscle pattern is, for the most part, programmed non-cell-autonomously. Connective tissues depend upon both muscle and skeletal tissues for their proper survival and development. Here, we employed a novel approach to dissect the coordination of musculoskeletal patterning during mouse limb development. Using both conditional gain- and loss-of-function approaches, we selectively deleted or activated the LIM-homeodomain transcription factor Lmx1b in skeletal progenitors using a Sox9-Cre knock-in allele. As Lmx1b is both necessary and sufficient to specify dorsal pattern, this approach allowed us to investigate the effect of selectively deleting or activating Lmx1b in skeletal progenitors on muscle, connective and skeletal tissues during limb development. Our results indicate that whereas Lmx1b activity is required autonomously in skeletal progenitors to direct dorsal pattern, loss or gain of Lmx1b activity in skeletal progenitors has no effect on muscle or connective tissue patterning. Hence, we show for the first time that skeletal and connective tissue patterning can be uncoupled, indicating a degree of autonomy in the formation of the musculoskeletal system.