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Showing papers on "Regulation of gene expression published in 1996"


Journal ArticleDOI
TL;DR: HIF-1 is implicate in the activation of VEGF transcription in hypoxic cells and this work demonstrates the involvement of Hif-1 in theactivation of V EGF transcription.
Abstract: Expression of vascular endothelial growth factor (VEGF) is induced in cells exposed to hypoxia or ischemia. Neovascularization stimulated by VEGF occurs in several important clinical contexts, including myocardial ischemia, retinal disease, and tumor growth. Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric basic helix-loop-helix protein that activates transcription of the human erythropoietin gene in hypoxic cells. Here we demonstrate the involvement of HIF-1 in the activation of VEGF transcription. VEGF 5'-flanking sequences mediated transcriptional activation of reporter gene expression in hypoxic Hep3B cells. A 47-bp sequence located 985 to 939 bp 5' to the VEGF transcription initiation site mediated hypoxia-inducible reporter gene expression directed by a simian virus 40 promoter element that was otherwise minimally responsive to hypoxia. When reporters containing VEGF sequences, in the context of the native VEGF or heterologous simian virus 40 promoter, were cotransfected with expression vectors encoding HIF-1alpha and HIF-1beta (ARNT [aryl hydrocarbon receptor nuclear translocator]), reporter gene transcription was much greater in both hypoxic and nonhypoxic cells than in cells transfected with the reporter alone. A HIF-1 binding site was demonstrated in the 47-bp hypoxia response element, and a 3-bp substitution eliminated the ability of the element to bind HIF-1 and to activate transcription in response to hypoxia and/or recombinant HIF-1. Cotransfection of cells with an expression vector encoding a dominant negative form of HIF-1alpha inhibited the activation of reporter transcription in hypoxic cells in a dose-dependent manner. VEGF mRNA was not induced by hypoxia in mutant cells that do not express the HIF-1beta (ARNT) subunit. These findings implicate HIF-1 in the activation of VEGF transcription in hypoxic cells.

3,709 citations


Journal ArticleDOI
TL;DR: The tissue-specific expression of a putative secreted protein suggests that this factor may function as a novel signaling molecule for adipose tissue.

2,270 citations


Journal ArticleDOI
TL;DR: The efficacy of different antioxidants to favorably influence the molecular mechanisms implicated in human disease should be a critical determinant of its selection for clinical studies.
Abstract: Reactive oxygen species (ROS) are implicated in the pathogenesis of a wide variety of human diseases. Recent evidence suggests that at moderately high concentrations, certain forms of ROS such as H202 may act as signal transduction messengers. To develop a better understanding of the exact mechanisms that underlie ROS-dependent disorders in biological systems, recent studies have investigated the regulation of gene expression by oxidants, antioxidants, and other determinants of the intracellular reduction-oxidation (redox) state. At least two well-defined transcription factors, nuclear factor (NF) kappa B and activator protein (AP) -1 have been identified to be regulated by the intracellular redox state. The regulation of gene expression by oxidants, antioxidants, and the redox state has emerged as a novel subdiscipline in molecular biology that has promising therapeutic implications. Binding sites of the redox-regulated transcription factors NF-kappa B and AP-1 are located in the promoter region of a large variety of genes that are directly involved in the pathogenesis of diseases, e.g., AIDS, cancer, atherosclerosis and diabetic complications. Biochemical and clinical studies have indicated that antioxidant therapy may be useful in the treatment of disease. Critical steps in the signal transduction cascade are sensitive to oxidants and antioxidants. Many basic events of cell regulation such as protein phosphorylation and binding of transcription factors to consensus sites on DNA are driven by physiological oxidant-antioxidant homeostasis, especially by the thiol-disulfide balance. Endogenous glutathione and thioredoxin systems, and the exogenous lipoate-dihydrolipoate couple may therefore be considered to be effective regulators of redox-sensitive gene expression. The efficacy of different antioxidants to favorably influence the molecular mechanisms implicated in human disease should be a critical determinant of its selection for clinical studies.

1,975 citations


Journal ArticleDOI
27 Dec 1996-Cell
TL;DR: The method to create mice in which the deletion of virtually any gene of interest is restricted to a subregion or a specific cell type in the brain such as the pyramidal cells of the hippocampal CA1 region should allow a more precise analysis of the impact of a gene mutation on animal behaviors.

1,226 citations


Journal ArticleDOI
TL;DR: A set of plasmids has been constructed utilizing the promoter, 5′ untranslated exon, and first intron of the maize ubiquitin (Ubi-1) gene to drive expression of protein coding sequences of choice to provide expression of biotechnologically important protein products in transgenic plants.
Abstract: A set of plasmids has been constructed utilizing the promoter, 5' untranslated exon, and first intron of the maize ubiquitin (Ubi-1) gene to drive expression of protein coding sequences of choice. Plasmids containing chimaeric genes for ubiquitin-luciferase (Ubi-Luc), ubiquitin-beta-glucuronidase (Ubi-GUS), and ubiquitin-phosphinothricin acetyl transferase (Ubi-bar) have been generated, as well as a construct containing chimaeric genes for both Ubi-GUS and Ubi-bar in a single plasmid. Another construct was generated to allow cloning of protein coding sequences of choice on Bam HI and Bam HI-compatible restriction fragments downstream of the Ubi-1 gene fragment. Because the Ubi-1 promoter has been shown to be highly active in monocots, these constructs may be useful for generating high-level gene expression of selectable markers to facilitate efficient transformation of monocots, to drive expression of reference reporter genes in studies of gene expression, and to provide expression of biotechnologically important protein products in transgenic plants.

1,224 citations


Journal ArticleDOI
19 Jul 1996-Science
TL;DR: Although ISS are necessary for gene vaccination, they down-regulate gene expression and thus may interfere with gene replacement therapy by inducing proinflammatory cytokines.
Abstract: Vaccination with naked DNA elicits cellular and humoral immune responses that have a T helper cell type 1 bias. However, plasmid vectors expressing large amounts of gene product do not necessarily induce immune responses to the encoded antigens. Instead, the immunogenicity of plasmid DNA (pDNA) requires short immunostimulatory DNA sequences (ISS) that contain a CpG dinucleotide in a particular base context. Human monocytes transfected with pDNA or double-stranded oligonucleotides containing the ISS, but not those transfected with ISS-deficient pDNA or oligonucleotides, transcribed large amounts of interferon-α, interferon-β, and interleukin-12. Although ISS are necessary for gene vaccination, they down-regulate gene expression and thus may interfere with gene replacement therapy by inducing proinflammatory cytokines.

1,198 citations


Journal ArticleDOI
TL;DR: In situ hybridization showed that the T/ebp gene is expressed in the normal thyroid, lung bronchial epithelium, and specific areas of the forebrain during early embryogenesis, establishing that the expression of T/EBP, a transcription factor known to control thyroid-specific gene transcription, is also essential for organogenesis of the thyroid, lungs, ventral forebrain, and pituitary.
Abstract: The thyroid-specific enhancer-binding protein (T/ebp) gene was disrupted by homologous recombination in embryonic stem cells to generate mice lacking T/EBP expression. Heterozygous animals developed normally, whereas mice homozygous for the disrupted gene were born dead and lacked the lung parenchyma. Instead, they had a rudimentary bronchial tree associated with an abnormal epithelium in their pleural cavities. Furthermore, the homozygous mice had no thyroid gland but had a normal parathyroid. In addition, extensive defects were found in the brain of the homozygous mice, especially in the ventral region of the forebrain. The entire pituitary, including the anterior, intermediate, and posterior pituitary, was also missing. In situ hybridization showed that the T/ebp gene is expressed in the normal thyroid, lung bronchial epithelium, and specific areas of the forebrain during early embryogenesis. These results establish that the expression of T/EBP, a transcription factor known to control thyroid-specific gene transcription, is also essential for organogenesis of the thyroid, lung, ventral forebrain, and pituitary.

1,147 citations


Journal ArticleDOI
27 Dec 1996-Cell
TL;DR: Two important Ca2+/calmodulin (CaM)-regulated mechanisms in hippocampal neurons are found: a CaM kinase cascade involving nuclear CaMKIV and a calcineurin-dependent regulation of nuclear protein phosphatase 1 activity.

1,112 citations


Journal ArticleDOI
TL;DR: It is reported here that Jun, Fos, Fra, and NRF nuclear transcription factors bind to the hARE, indicating that hARE-mediated expression of the NQO1 gene and its induction by xenobiotics and antioxidants are mediated by Nrf1 and Nrf2.
Abstract: Twenty-four base pairs of the human antioxidant response element (hARE) are required for high basal transcription of the NAD(P)H:quinone oxidoreductase1 (NQO1) gene and its induction in response to xenobiotics and antioxidants. hARE is a unique cis-element that contains one perfect and one imperfect AP1 element arranged as inverse repeats separated by 3 bp, followed by a “GC” box. We report here that Jun, Fos, Fra, and Nrf nuclear transcription factors bind to the hARE. Overexpression of cDNA derived combinations of the nuclear proteins Jun and Fos or Jun and Fra1 repressed hARE-mediated chloramphenicol acetyltransferase (CAT) gene expression in transfected human hepatoblastoma (Hep-G2) cells. Further experiments suggested that this repression was due to overexpression of c-Fos and Fra1, but not due to Jun proteins. The Jun (c-Jun, Jun-B, and Jun-D) proteins in all the possible combinations were more or less ineffective in repression or upregulation of hARE-mediated gene expression. Interestingly, overexpression of Nrf1 and Nrf2 individually in Hep-G2 and monkey kidney (COS1) cells significantly increased CAT gene expression from reporter plasmid hARE-thymidine kinase-CAT in transfected cells that were inducible by β-naphthoflavone and tert-butyl hydroquinone. These results indicated that hARE-mediated expression of the NQO1 gene and its induction by xenobiotics and antioxidants are mediated by Nrf1 and Nrf2. The hARE-mediated basal expression, however, is repressed by overexpression of c-Fos and Fra1.

1,047 citations


Journal ArticleDOI
TL;DR: It is demonstrated using Northern analysis that treatment of various cell lines with IL-6 for 6-48 h results in a significant induction of VEGF mRNA, and it is shown that the 5′-UTR is important for the expression of V EGF.

1,045 citations


Journal ArticleDOI
01 Nov 1996-Cell
TL;DR: It is proposed that the complex regulation of Hac1p expression serves to provide multiple levels at which the UPR can be controlled.

Journal ArticleDOI
05 Sep 1996-Nature
TL;DR: In vitro and in vivo evidence is provided that identifies the CREB-binding protein (CBP) and its homologue P300 as cofactors mediating nuclear-receptor-activated gene transcription and may serve as integrators of extracellular and intracellular signalling pathways.
Abstract: The nuclear receptor superfamily includes receptors for steroids, retinoids, thyroid hormone and vitamin D, as well as many related proteins. An important feature of the action of the lipophilic hormones and vitamins is that the maintenance of homeostatic function requires both intrinsic positive and negative regulation. Here we provide in vitro and in vivo evidence that identifies the CREB-binding protein (CBP) and its homologue P300 (refs 6,7) as cofactors mediating nuclear-receptor-activated gene transcription. The role of CBP/P300 in the transcriptional response to cyclic AMP, phorbol esters, serum, the lipophilic hormones and as the target of the E1A oncoprotein suggests they may serve as integrators of extracellular and intracellular signalling pathways.

Journal ArticleDOI
TL;DR: It is reported here that conditional site-specific recombination can be achieved in mice using a new version of the Cre/lox system, and excision of a chromosomally integrated gene flanked by loxP sites can be induced by administration of tamoxifen to these transgenic mice, whereas no excision could be detected in untreated animals.
Abstract: Current mouse gene targeting technology is unable to introduce somatic mutations at a chosen time and/or in a given tissue. We report here that conditional site-specific recombination can be achieved in mice using a new version of the Cre/lox system. The Cre recombinase has been fused to a mutated ligand-binding domain of the human estrogen receptor (ER) resulting in a tamoxifen-dependent Cre recombinase, Cre-ERT, which is activated by tamoxifen, but not by estradiol. Transgenic mice were generated expressing Cre-ERT under the control of a cytomegalovirus promoter. We show that excision of a chromosomally integrated gene flanked by loxP sites can be induced by administration of tamoxifen to these transgenic mice, whereas no excision could be detected in untreated animals. This conditional site-specific recombination system should allow the analysis of knockout phenotypes that cannot be addressed by conventional gene targeting.

Book
01 Jan 1996
TL;DR: Historical overview of epigenetic mechanisms DNA methylation paramutation, imprinting and X inactivation repeated genes and gene silencing nuclear organization and chromatin structure transposable elements and viruses.
Abstract: Historical overview of epigenetic mechanisms DNA methylation paramutation, imprinting and X inactivation repeated genes and gene silencing nuclear organization and chromatin structure transposable elements and viruses. Appendices.

Journal ArticleDOI
TL;DR: The results indicate that cDNA-AFLP is a broadly applicable technology for identifying developmentally regulated genes and rapid and simple verification of band identity may be achieved.
Abstract: Using a highly synchronous in vitro tuberization system, in combination with an amplified restriction fragment polymorphism (AFLP)-derived technique for RNA fingerprinting (cDNA-AFLP), transcriptional changes at and around the time point of potato tuberization have been analyzed. The targeted expression analysis of a specific transcript coding for the major potato storage protein, patatin and a second transcript, coding for ADP-glucose pyrophosphorylase, a key gene in the starch biosynthetic pathway is described. This paper confirms that kinetics of expression revealed by cDNA-AFLP analysis are comparable to those found in Northern analysis. Furthermore, this paper reports the isolation and analysis of two tuber-specific transcript-derived fragments (TDFs) coding for the lipoxygenase enzyme, which are differentially induced around the time point of tuber formation. Analysis of the two lox TDFs demonstrates that it is possible to dissect the expression modalities of individual transcripts, not independently detectable by Northern analysis. Finally, it is shown that using cDNA-AFLP, rapid and simple verification of band identity may be achieved. The results indicate that cDNA-AFLP is a broadly applicable technology for identifying developmentally regulated genes.

Journal ArticleDOI
TL;DR: The kinetics, control, and efficiency of nisin-induced expression directed by the nisA promoter region were studied in Lactococcus lactis with transcriptional and translational fusions to the gusA reporter genes.
Abstract: The kinetics, control, and efficiency of nisin-induced expression directed by the nisA promoter region were studied in Lactococcus lactis with transcriptional and translational fusions to the gusA reporter genes. In the nisin-producing L. lactis strain NZ9700, the specific beta-glucuronidase activity increased very rapidly after mid-exponential growth until the maximum level at the start of the stationary phase was reached. Expression of the gusA gene was also studied in L. lactis NZ9800, an NZ9700 derivative carrying a deletion in the structural nisA gene that abolishes nisin production, and in L. lactis NZ3900, an MG1363 derivative containing the regulatory nisRK genes integrated in the chromosome. In both strains, beta-glucuronidase activity was linearly dependent on the amount of nisin added to the medium. Without nisin, no beta-glucuronidase production was observed. To optimize translation initiation, an expression vector was constructed by fusing the gusA gene translationally to the start codon of the nisA gene. Use of the translational fusion vector yielded up to six times more beta-glucuronidase activity than the transcriptional fusion vector in these strains after induction by nisin. In this way, gene expression can be achieved in a dynamic range of more than 1,000-fold. The beta-glucuronidase activity was found to be up to 25-fold higher in extracts of strain NZ3900 than in extracts of strain NZ9800. This translational fusion vector was used for high-level production of aminopeptidase N, up to 47% of the total intracellular protein. These results clearly illustrate the potential of the nisin-inducible expression system for overproduction of desired proteins.

Journal ArticleDOI
04 Oct 1996-Cell
TL;DR: A novel, NeuroD-related bHLH protein, NEUROGENIN, whose expression precedes that of NeuroD in both mouse and Xenopus is described, suggesting that it functions as a vertebrate neuronal determination factor.

Journal ArticleDOI
TL;DR: It is demonstrated that the transcription factors serum response factor (SRF) and Elk-1 can mediate glutamate induction of transcription through the SRE in cortical neurons, suggesting that SRF, Elk, and ERKs may have important roles in neuroplasticity.
Abstract: The regulation of gene expression by neurotransmitters is likely to play a key role in neuroplasticity both during development and in the adult animal. Therefore, it is important to determine the mechanisms of neuronal gene regulation to understand fully the mechanisms of learning, memory, and other long-term adaptive changes in neurons. The neurotransmitter glutamate stimulates rapid and transient induction of many genes, including the c-fos proto-oncogene. The c-fos promoter contains several critical regulatory elements, including the serum response element (SRE), that mediate glutamate-induced transcription in neurons; however, the mechanism by which the SRE functions in neurons has not been defined. In this study, we sought to identify transcription factors that mediate glutamate induction of transcription through the SRE in cortical neurons and to elucidate the mechanism(s) of transcriptional activation by these factors. To facilitate this analysis, we developed an improved calcium phosphate coprecipitation procedure to transiently introduce DNA into primary neurons, both efficiently and consistently. Using this protocol, we demonstrate that the transcription factors serum response factor (SRF) and Elk-1 can mediate glutamate induction of transcription through the SRE in cortical neurons. There are at least two distinct pathways by which glutamate signals through the SRE: an SRF-dependent pathway that can operate in the absence of Elk and an Elk-dependent pathway. Activation of the Elk-dependent pathway of transcription seems to require phosphorylation of Elk-1 by extracellular signal-regulated kinases (ERKs), providing evidence for a physiological function of ERKs in glutamate signaling in neurons. Taken together, these findings suggest that SRF, Elk, and ERKs may have important roles in neuroplasticity.


Journal ArticleDOI
TL;DR: Both tetracycline-controlled transcriptional activation systems provide genetic switches that permit the quantitative control of gene activities in transgenic mice in a tissue-specific manner and, thus, suggest possibilities for the generation of a novel type of conditional mutants.
Abstract: The tet regulatory system in which doxycycline (dox) acts as an inducer of specifically engineered RNA polymerase II promoters was transferred into transgenic mice. Tight control and a broad range of regulation spanning up to five orders of magnitude were monitored dependent on the dox concentration in the water supply of the animals. Administration of dox rapidly induces the synthesis of the indicator enzyme luciferase whose activity rises over several orders of magnitude within the first 4 h in some organs. Induction is complete after 24 h in most organs analyzed. A comparable regulatory potential was revealed with the tet regulatory system where dox prevents transcription activation. Directing the synthesis of the tetracycline-controlled transactivator (tTA) to the liver led to highly specific regulation in hepatocytes where, in presence of dox, less than one molecule of luciferase was detected per cell. By contrast, a more than 10(5)-fold activation of the luciferase gene was observed in the absence of the antibiotic. This regulation was homogeneous throughout but stringently restricted to hepatocytes. These results demonstrate that both tetracycline-controlled transcriptional activation systems provide genetic switches that permit the quantitative control of gene activities in transgenic mice in a tissue-specific manner and, thus, suggest possibilities for the generation of a novel type of conditional mutants.

Journal ArticleDOI
TL;DR: Activated NF-kappaB was detected in the fibrotic-thickened intima/media and atheromatous areas of the atherosclerotic lesion and electrophoretic mobility shift assays and colocalization of activated NF- kappaB with NF-KappaB target gene expression suggest functional implications for this transcription factor in the atherological lesion.
Abstract: Nuclear factor-kappa B (NF-kappaB)/Rel transcription factors play an important role in the inducible regulation of a variety of genes involved in the inflammatory and proliferative responses of cells. The present study was designed to elucidate the implication of NF-kappaB/Rel in the pathogenesis of atherosclerosis. Activation of the dimeric NF-kappaB complex is regulated at a posttranslational level and requires the release of the inhibitor protein IkappaB. The newly developed mAb alpha-p65mAb recognizes the IkappaB binding region on the p65 (RelA) DNA binding subunit and therefore selectively reacts with p65 in activated NF-kappaB. Using immunofluorescence and immunohistochemical techniques, activated NF-kappaB was detected in the fibrotic-thickened intima/media and atheromatous areas of the atherosclerotic lesion. Activation of NF-kappaB was identified in smooth muscle cells, macrophages, and endothelial cells. Little or no activated NF-kappaB was detected in vessels lacking atherosclerosis. Electrophoretic mobility shift assays and colocalization of activated NF-kappaB with NF-kappaB target gene expression suggest functional implications for this transcription factor in the atherosclerotic lesion. This study demonstrates the presence of activated NF-kappaB in human atherosclerotic tissue for the first time. Atherosclerosis, characterized by features of chronic inflammation and proliferative processes, may be a paradigm for the involvement of NF-kappaB/Rel in chronic inflammatory disease.

Journal ArticleDOI
TL;DR: The hypothesis that HA fragments generated during inflammation induce the expression of macrophage genes which are important in the development and maintenance of the inflammatory response is supported.
Abstract: Hyaluronan (HA) is a glycosaminoglycan constituent of extracellular matrix In its native form HA exists as a high molecular weight polymer, but during inflammation lower molecular weight fragments accumulate We have identified a collection of inflammatory genes induced in macrophages by HA fragments but not by high molecular weight HA These include several members of the chemokine gene family: macrophage inflammatory protein-1alpha, macrophage inflammatory protein-1beta, cytokine responsive gene-2, monocyte chemoattractant protein-1, and regulated on activation, normal T cell expressed and secreted HA fragments as small as hexamers are capable of inducing expression of these genes in a mouse alveolar macrophage cell line, and monoclonal antibody to the HA receptor CD44 completely blocks binding of fluorescein-labeled HA to these cells and significantly inhibits HA-induced gene expression We also investigated the ability of HA fragments to induce chemokine gene expression in human alveolar macrophages from patients with idiopathic pulmonary fibrosis and found that interleukin-8 mRNA is markedly induced These data support the hypothesis that HA fragments generated during inflammation induce the expression of macrophage genes which are important in the development and maintenance of the inflammatory response

Journal ArticleDOI
TL;DR: It is shown that p16 transcription is affected by the status of pRB and a region in the p16 promoter that is required for this response, but the effect is not sufficient to account for the differences in p16 RNA levels between pRB-positive and -negative cells.
Abstract: p16CDKN2 specifically binds to and inhibits the cyclin-dependent kinases CDK4 and CDK6, which function as regulators of cell cycle progression in G1 by contributing to the phosphorylation of the retinoblastoma protein (pRB). Human cell lines lacking functional pRB contain high levels of p16 RNA and protein, suggesting a negative feedback loop by which pRB might regulate p16 expression in late G1. By a combination of nuclear run-on assays and promoter analyses in human fibroblasts expressing a temperature-sensitive simian virus 40 T antigen, we show that p16 transcription is affected by the status of pRB and define a region in the p16 promoter that is required for this response. However, the effect is not sufficient to account for the differences in p16 RNA levels between pRB-positive and -negative cells. Moreover, p16 RNA is extremely stable, and the levels do not change appreciably during the cell cycle. Primary human fibroblasts express very low levels of p16, but the RNA and protein accumulate in late-passage, senescent cells. The apparent overexpression of p16 in pRB-negative cell lines is therefore caused by at least two factors: loss of repression by pRB and an increase in the number of population doublings.

Journal ArticleDOI
TL;DR: In this paper, an emerging topic of great interest is the basis for specificity in the activation of individual MAPKs and their ability to recognize their substrates, and many tiers in the regulation of the activities of MAPK, as well as different routes that lead to the activation.

Journal ArticleDOI
17 May 1996-Cell
TL;DR: Although overexpression of E2F-1 in tissue culture cells can stimulate cell proliferation and be oncogenic, loss of E 2F- 1 in mice results in tumorigenesis, demonstrating that E2f-1 also functions as a tumor suppressor.

Journal ArticleDOI
TL;DR: This review focuses on recent advances on early auxin-inducible gene expression and possible functions of the polypeptides encoded, which are likely candidates to play a pivotal role in mediating growth-stimulating effects of the hormone.
Abstract: The plant hormone IAA (or auxin) is central to the control of plant growth and development. Processes governed by auxin in concert with other plant growth regulators include development of vascular tissues, formation of lateral and adventitious roots, control of apical dominance, and tropic responses (Went and Thimann, 1937). At the level of cellular physiology, auxin profoundly affects turgor, elongation, division, and cell differentiation, the major driving and shaping forces in morphogenesis and oncogenesis. The molecular mechanisms of auxin action are still unknown, although it is now well established that auxin modulates membrane function and gene expression (for review, see Napier and Venis, 1995). These biochemical changes, in turn, most likely affect fundamental aspects of plant morphology and physiology. However, a causal relationship between auxin-mediated alterations in gene expression or membrane function and a particular growth process has not yet been demonstrated. Despite its critical role in plant development and the immense volume of studies on the diverse auxin effects, understanding of the molecular mechanisms of auxin action remains one of the major challenges in plant biology. The signal transduction cascades leading from auxin perception to altered gene expression or membrane function hold the key in our attempts to elucidate the primary mechanism(s) of auxin action. An array of experimental strategies has been mounted to investigate auxin signaling pathways. The combination of biochemical, molecular, and genetic approaches will allow for significant new insights into how the hormone works in molecular terms (Fig. 1). One strategy employs genetics and reverse genetics to construct transgenic plants with perturbations in auxin homeostasis and to screen for mutants with defects in auxinrelated physiology. Transgenic plants expressing altered hormone levels have already resolved some longstanding questions in plant physiology. Mutant plants defective in auxin responses will rejuvenate and stimulate research by identifying novel genes involved in hormone perception, signal transduction, and physiological responses (for review, see Hobbie and Estelle, 1994; Klee and Romano, 1994). The first significant result (to our knowledge) of this approach was the cloning of the AXR1 gene, which encodes a protein related to the ubiquitin-activating enzyme El (Leyser et al., 1993). Although AXRl is probably not a functional El homolog, it is nonetheless an exquisite example of the potential of molecular genetics to connect the unexpected. The biochemical strategy is based on the identification of auxin receptors and subsequent isolation of interacting components. The search for auxin receptors has led to the discovery of a number of soluble and membranebound proteins that bind auxin with moderate but physiologically relevant affinity. Their functional role in auxin signaling is still unclear and is a major target of current research (for review, see Jones, 1994; Napier and Venis, 1995). Auxin-regulated genes provide yet another source of molecular tools to dissect auxin action. The hormone modulates gene expression in a wide variety of plant tissues and cell types over a broad period of time (for review, see Guilfoyle, 1986; Theologis, 1986). However, early genes selectively induced as a primary response to auxin and prior to the initiation of cell growth are likely candidates to play a pivotal role in mediating growth-stimulating effects of the hormone. This review focuses on recent advances in our knowledge on early auxin-inducible gene expression and possible functions of the polypeptides encoded.

Journal ArticleDOI
TL;DR: The cloned ER gene encodes a putative receptor protein kinases, and the results suggest that cell-cell communication mediated by a receptor kinase has an important role in plant morphogenesis.
Abstract: Arabidopsis Landsberg erecta is one of the most popular ecotypes and is used widely for both molecular and genetic studies. It harbors the erecta (er) mutation, which confers a compact inflorescence, blunt fruits, and short petioles. We have identified five er mutant alleles from ecotypes Columbia and Wassilewskija. Phenotypic characterization of the mutant alleles suggests a role for the ER gene in regulating the shape of organs originating from the shoot apical meristem. We cloned the ER gene, and here, we report that it encodes a putative receptor protein kinases. The deduced ER protein contains a cytoplasmic protein kinase catalytic domain, a transmembrane region, and an extracellular domain consisting of leucine-rich repeats, which are thought to interact with other macromolecules. Our results suggest that cell-cell communication mediated by a receptor kinase has an important role in plant morphogenesis.

Journal ArticleDOI
TL;DR: The conservation of sexually dimorphic expression in two vertebrate classes which have significant differences in their sex determination mechanisms, points to a fundamental role for SOX9 in testis determination in vertebrates.
Abstract: Mutation analyses of patients with campomelic dysplasia, a bone dysmorphology and XY sex reversal syndrome, indicate that the SRY-related gene SOX9 is involved in both skeletal development and sex determination. To clarify the role SOX9 plays in vertebrate sex determination, we have investigated its expression during gonad development in mouse and chicken embryos. In the mouse, high levels of Sox9 mRNA were found in male (XY) but not female (XX) genital ridges, and were localised to the sex cords of the developing testis. Purified fetal germ cells lacked Sox9 expression, indicating that Sox9 expression is specific to the Sertoli cell lineage. Sex specificity of SOX9 protein expression was confirmed using a polyclonal antiserum. The timing and cell-type specificity of Sox9 expression suggests that Sox9 may be directly regulated by SRY. Male-specific expression of cSOX9 mRNA during the sex determination period was also observed in chicken genital ridges. The conservation of sexually dimorphic expression in two vertebrate classes which have significant differences in their sex determination mechanisms, points to a fundamental role for SOX9 in testis determination in vertebrates. Sox9 expression was maintained in the mouse testis during fetal and adult life, but no expression was seen at any stage by in situ hybridisation in the developing ovary. Male-specific expression was also observed in the cells surrounding the Mullerian ducts and in the epididymis, and expression in both sexes was detected in the developing collecting ducts of the metanephric kidney. These results suggest that SOX9 may have a wider role in the development of the genitourinary system.

Journal ArticleDOI
TL;DR: Examination of the developing testis in different genetic backgrounds suggests that Dhh regulates both early and late stages of spermatogenesis, and loss of Patched expression in Dhh mutants suggests a conservation in the Hedgehog signaling pathway between flies and mice, and indicates that Leydig cells may be the direct target of Dhh signaling.

Journal ArticleDOI
24 Oct 1996-Nature
TL;DR: A model is proposed to explain how TGF-β superfamily signals might regulate the expression of specific genes in the early embryo.
Abstract: The transforming-growth-factor-beta (TGF-beta) superfamily is critical for establishing mesoderm during early embryogenesis in Xenopus. The transcriptional activation of Mix.2, an immediate-early response gene specific to activin-like members of the TGF-beta superfamily, is associated with the rapid appearance of a site-specific DNA-binding activity that recognizes a fifty-base-pair regulatory element known as ARE in the Mix.2 promoter. Cloning of the site-specific DNA-binding component of this activity revealed it to be a new winged-helix transcription factor and a direct target for signalling by the TGF-beta superfamily. XMAD2, a recently identified TGF-beta signal transducer, forms a complex with the transcription factor in an activin-dependent fashion to generate an activated ARE-binding complex. A model is proposed to explain how TGF-beta superfamily signals might regulate the expression of specific genes in the early embryo.