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At least 73 records · Page 4Linked to original sources
PubMed1998

crooked legs encodes a family of zinc finger proteins required for leg morphogenesis and ecdysone-regulated gene expression during Drosophila metamorphosis.

Drosophila imaginal discs undergo extensive pattern formation during larval development, resulting in each cell acquiring a specific adult fate. The final manifestation of this pattern into adult structures is dependent on pulses of the steroid hormone ecdysone during metamorphosis, which trigger disc eversion, elongation and differentiation. We have defined genetic criteria that allow us to screen for ecdysone-inducible regulatory genes that are required for this transformation from patterned disc to adult structure. We describe here the first genetic locus isolated using these criteria: crooked legs (crol). crol mutants die during pupal development with defects in adult head eversion and leg morphogenesis. The crol gene is induced by ecdysone during the onset of metamorphosis and encodes at least three protein isoforms that contain 12-18 C2H2 zinc fingers. Consistent with this sequence motif, crol mutations have stage-specific effects on ecdysone-regulated gene expression. The EcR ecdysone receptor, and the BR-C, E74 and E75 early regulatory genes, are submaximally induced in crol mutants in response to the prepupal ecdysone pulse. These changes in gene activity are consistent with the crol lethal phenotypes and provide a basis for understanding the molecular mechanisms of crol action. The genetic criteria described here provide a new direction for identifying regulators of adult tissue development during insect metamorphosis.

Amino Acid Sequence↗
PubMed1989

FNR-dependent repression of the ndh gene of Escherichia coli and metal ion requirement for FNR-regulated gene expression.

The ndh gene of Escherichia coli which encodes an NADH dehydrogenase contains a putative FNR-binding site in its upstream non-coding region, and its expression has been investigated using an ndh-lacZ fusion. Expression of the fusion was found to be reduced during anaerobic growth, and experiments with hosts containing an fnr mutation and/or a multicopy fnr+ plasmid indicated that the anaerobic repression of the ndh gene is mediated by the FNR protein. Thus FNR can function as an anaerobic repressor as well as an anaerobic transcriptional activator. The results are consistent with the FNR-binding function attributed to the proposed consensus sequence. Using frdA- and ndh-lacZ fusions exhibiting positive and negative regulation by FNR, it was further shown that the depletion of metal ions in growth media with chelating agents mimics oxygen with respect to the activity of FNR. Possible roles for metal ions in the oxygen-sensing pathway associated with FNR function are discussed.

Amino Acid Sequence↗
PubMed1989

Receptor-mediated transport of heme by hemopexin regulates gene expression in mammalian cells.

Hemopexin (HPX) transports heme to liver parenchymal cells, undergoes receptor-mediated endocytosis, and recycles intact. Incubation of mouse hepatoma (Hepa) cells with heme-HPX causes a rapid dose- and time-dependent increase in the steady-state level of heme oxygenase (HO) mRNA. A maximum induction of 20-25-fold is achieved within 3 h after incubation with 10 microM heme-HPX. This accumulation of HO mRNA results primarily from increased transcription of the HO gene as judged by in vitro nuclear run-on assays. In addition, receptor-mediated transport of heme into Hepa cells significantly decreases the steady-state level of transferrin receptor (TfR) mRNA. While a 25-30-fold decrease in the amount of TfR mRNA is observed within 3 h of incubation of Hepa cells with 10 microM heme-HPX, no significant change in the rate of TfR gene transcription was detected. These regulatory effects of heme-HPX are not restricted to hepatic cells but are also observed in human promyelocytic HL-60 cells. This is the first direct demonstration of receptor-mediated transport of heme by hemopexin regulating gene expression in mammalian cells.

Animals↗
PubMed2005

Evaluation of the tetracycline promoter system for regulated gene expression in Kluyveromyces marxianus.

A tetracycline repressible promoter system designed for Saccharomyces cerevisiae was evaluated for use in Kluyveromyces marxianus. A plasmid was constructed containing the Escherichia coli beta-glucuronidase (gus) gene cloned downstream of the yeast tet-off promoter, the tetR-VP16 activator protein gene, and the URA3 gene for selection. The tet-off promoter-gus construct was integrated into the chromosomal DNA and tested under varying growth conditions in complex medium. The repressors tetracycline and doxycycline were both found to be effective for inhibiting gene expression. Doxycycline levels of 0.5 microg/mL or greater were sufficient to nearly completely suppress Gus synthesis. For most transformants, the induction ratio was approximately 2,000-fold. The tet-off promoter was effective at 30, 37, and 42 degrees C, although the overall Gus activity was highest at 37 degrees C. During exponential growth, little product was formed; expression increased dramatically in late exponential and early stationary phase. The promoter thus shows promise for protein synthesis following cell growth. No inducer is required and the repressor is only needed to prevent expression during the seed culture.

Cloning, Molecular↗
PubMed1987

Metal ion regulated gene expression: use of a plastocyanin-less mutant of Chlamydomonas reinhardtii to study the Cu(II)-dependent expression of cytochrome c-552.

Ac-208, a plastocyanin-deficient strain of Chlamydomonas reinhardtii, lacks the mRNA for pre-apoplastocyanin although Southern analyses indicate no gross deletion in the single gene for pre-apoplastocyanin. The alternative electron carrier, cytochrome c-552, is expressed only in Cu(II)-deficient cells of the mutant. Thus, the mutant strain can grow photoautotrophically in Cu(II)-deficient but not in Cu(II)-sufficient medium. This indicates that the absence of plastocyanin is the only defect (with respect to photosynthesis) in ac-208. Phenotypic revertants of ac-208 are capable of photoautotrophic growth by virtue of constitutive [with respect to Cu(II)] synthesis of cytochrome c-552. The revertant phenotype is attributed to a decreased internal concentration of Cu(II). This indicates that the Cu(II)-dependent expression of cytochrome c-552 synthesis is affected directly by the concentration of Cu(II) rather than indirectly by the level of plastocyanin in the cell.

Chlamydomonas↗
PubMed1986

Cloning of a major developmentally regulated gene expressed in mature females of Schistosoma mansoni.

A cDNA library constructed from RNA isolated from adult Schistosoma mansoni has been screened by differential hybridization to identify clones corresponding to genes highly expressed by female worms. Several such cDNAs encoding the same highly abundant mRNA species were identified. Studies with one of these (pSF10) which contained a 500 base pair insert demonstrated that this gene was not expressed in immature females or eggs and encoded a polypeptide of approximately 35 000 daltons. Quantitation of the levels of RNA showed that 10% of the total RNA of female parasites was homologous to pSF10. A single gene corresponding to pSF10 was identified in Southern blotting experiments using adult worm DNA. The cloning of this gene will facilitate study of the molecular and genetic events controlling female schistosome maturation.

Animals↗
PubMed2002

Hypoxic induction of hypoxia-inducible factor-1alpha and oxygen-regulated gene expression in mitochondrial DNA-depleted HeLa cells.

Hypoxia increases the accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) and induces transcription of a variety of genes including vascular endothelial growth factor (VEGF) gene through oxygen sensing mechanisms. In one model, mitochondria-derived reactive oxygen species (ROS) are suggested to be involved in oxygen sensing. Here, we found that hypoxia increased ROS generation and VEGF gene expression in HeLa cells. To further investigate the role of ROS in oxygen sensing, we compared the hypoxic response between rho(0) HeLa cells lacking mitochondrial DNA (EB8) and rho(0) HeLa cells containing mitochondrial DNA from wild-type HeLa cells (HeEB1). The results showed that, although hypoxia markedly increased ROS generation in HeEB1 cells but not in EB8 cells, EB8 cells showed essentially a normal response to hypoxia, as assessed by VEGF gene promoter activity, HIF-1alpha accumulation, and HIF-1 target gene expressions. These results indicate that mitochondria-derived ROS generated in response to hypoxia are not necessary for oxygen sensing in HeLa cells.

Cell Hypoxia↗
PubMed2001

Repression of androgen-regulated gene expression by dominant negative androgen receptors.

The androgen receptor (AR) is a ligand-dependent transcription activator responsible for male sexual development. In order to specifically inhibit the AR pathway, dominant negative ARs were constructed by inactivation of the major transactivation domains of the wild type AR and fusing this mutant (AR122) to the Krüppel-associated box (KRAB) repressor domain and/or histone deacetylase (HDAC1). The HDAC1-KRAB-AR122 protein was the most successful dominant negative AR, capable of repressing the wild type AR ninefold when co-expressed at a 1:1 plasmid ratio. A maximal repression of 41-fold was achieved when HDAC1-KRAB-AR122 was cotransfected with the wild type AR at a 4:1 plasmid ratio. HDAC1-KRAB-AR122 repressed transcription in a ligand-dependent manner since it inhibited a constitutively active AR mutant (AR5) only in the presence of agonists. High concentrations of partial agonists such as RU486, cyproterone acetate, and estradiol were also capable of triggering repression by HDAC1-KRAB-AR122. The potent dominant negative AR proteins might prove useful tools to inhibit AR function in vitro and in vivo.

Animals↗
PubMed2003

Overexpressed glucocorticoid receptor negatively regulates gene expression under conditions that favour accumulation of non-hormone-binding forms of the receptor.

Previous reports have suggested that the native hormone-responsive glucocorticoid receptor is a heterocomplex with hsp90 and that the receptor constantly cycles between the hormone-responsive and an inactive state, with complex assembly and turnover being driven by hsp70 and hsp90, respectively. Since hsp70 appears to be titrated in cells that transiently overexpress the receptor, assembly intermediates may accumulate when more receptor is produced than can be assembled to hormone-responsive complex. Comparison of receptor protein and hormone-binding levels in extracts from transiently transfected COS-7 cells revealed the presence of non-hormone-binding receptor forms in addition to the native heterocomplex. The receptor was predominantly nuclear in the majority of the transfected cells even in the absence of hormone, with the DNA-binding domain (DBD) being necessary for nuclear localisation. Moreover, the unliganded receptor exhibited constitutive DNA-binding activity and reactivity towards antibodies against the hinge region where NLS1 is known to reside. By comparing fluorography to immunoblotting of two-dimensional SDS-PAGE of cross-linked [3H]dexamethasone-mesylate-labelled receptor, we detected non-hormone-binding receptor species capable of binding DNA in vitro. In addition, using a constitutively active receptor mutant, we found that the overexpressed wild-type receptor was capable of repressing mutant-activated transcription of transiently and stably transfected reporter genes alike in a DBD-dependent manner.

Animals↗
PubMed1998

The selectable marker neo gene down-regulates gene expression from retroviral vectors containing an internal ribosome entry site.

The internal ribosome entry site (IRES) from the picornavirus family has frequently been used to express multiple genes from a polycistronic message in retroviral vectors. While examining factors affecting levels of gene expression in IRES-containing retroviral vectors, it was found that retroviral vectors expressing the two genes linked by IRES, the reporter gene and the selectable marker neo, produced significantly lower levels of protein than those containing a reporter gene alone. This observation has been made with various cDNA sequences. However, when the neo was replaced with a different cDNA, the level of gene expression was increased, often to the level achieved with a vector expressing a single gene, suggesting that the bacterial neo sequence has a negative effect on expression. Analysis of the steady-state RNA levels isolated from transfected packaging cells showed that the neo-containing retroviral vectors produced significantly lower levels of RNA than those lacking this bacterial sequence indicating that neo interferes with expression of the neighboring gene at the level of RNA. Furthermore, the order of genes in the IRES-neo-containing vectors appeared to be more important than in the vector lacking the neo sequence. Our results suggest that neo has to be used in the retroviral vector with care, especially when a high level gene expression is needed.

Animals↗
PubMed1989

Analysis of rat prolactin promoter sequences that mediate pituitary-specific and 3',5'-cyclic adenosine monophosphate-regulated gene expression in vivo.

To determine the rat PRL (rPRL) promoter sequences that mediate pituitary-specific and cAMP-induced gene expression in vivo, various lengths of the rPRL promoter were ligated to the luciferase reporter gene and introduced into pituitary and non-pituitary cell lines. A 30-fold increase in rPRL promoter activity was observed in GH4 rat pituitary tumor cells compared to nonpituitary Rat2 fibroblast and HeLa cervical carcinoma cells. About 45% of this cell-specific promoter activity was competed by a plasmid containing the -67 to -45 rPRL promoter region, which is the most proximal binding site for a lactotroph-specific factor. Compared to a -425 rPRL construct, transfection with rPRL 5'-end points of -212, -178, and -127 contained 23%, 45%, and 1%, respectively, of luciferase activity. Forskolin stimulation resulted in a 10-fold induction of all the rPRL promoter fragments tested. Of note, a -127 deletion which was devoid of any basal promoter activity was also induced 10-fold by forskolin. The forskolin effect was abolished when GH4 rat pituitary cells were cotransfected with a plasmid encoding a protein kinase A inhibitor, indicating protein kinase A is involved in the activation mechanism. These data document that both positive and negative effectors influence basal rPRL promoter activity. Furthermore, the minimum sequences required for pituitary-specific rPRL promoter activity are altered by intracellular cAMP levels. Taken together, the data indicate that hormone-activated and cell-specific factors may interact to establish a particular setpoint for rPRL gene expression.

Animals↗
PubMed2006

Thyroid hormone receptors TRalpha1 and TRbeta differentially regulate gene expression of Kcnq4 and prestin during final differentiation of outer hair cells.

Thyroid hormone (TH or T3) and TH-receptor beta (TRbeta) have been reported to be relevant for cochlear development and hearing function. Mutations in the TRbeta gene result in deafness associated with resistance to TH syndrome. The effect of TRalpha1 on neither hearing function nor cochlear T3 target genes has been described to date. It is also uncertain whether TRalpha1 and TRbeta can act simultaneously on different target genes within a single cell. We focused on two concomitantly expressed outer hair cell genes, the potassium channel Kcnq4 and the motor protein prestin Slc26a5. In outer hair cells, TH enhanced the expression of the prestin gene through TRbeta. Simultaneously Kcnq4 expression was activated in the same cells by derepression of TRalpha1 aporeceptors mediated by an identified THresponse element, which modulates KCNQ4 promoter activity. We show that T3 target genes can differ in their sensitivity to TH receptors having the ligand either bound (holoreceptors) or not bound (aporeceptors) within single cells, and suggest a role for TRalpha1 in final cell differentiation.

Animals↗
PubMed1982

Developmentally regulated gene expression in Drosophila larval fat bodies.

During third-instar larval development of Drosophila melanogaster, the fat body tissue synthesizes six major methionine-containing polypeptides, three of which are the alpha, beta, and gamma subunits of the hexameric larval serum protein LSP-1, a fourth is the single subunit of the hexameric larval serum protein LSP-2, and the other two are polypeptides P6 and P1. Genomic DNA clones of the six structural genes for the polypeptides were isolated and characterized. Each gene maps by in situ hybridization at a single chromosomal site and appears to be present as a single copy in the genome. The LSP-1 and LSP-2 genes show striking regulatory similarities: The LSP-1 beta and gamma transcripts are first detected in fat bodies within an hour after the second molt, and the LSP-1 alpha and LSP-2 transcripts a few hours later; the four transcripts are subsequently maintained at high levels during most of the third instar and rapidly decrease shortly before pupariation. Ecdysterone increases the levels of at least three of the four LSP transcripts in the fat bodies when ecdysterone-deficient larvae from the temperature-sensitive mutant ecd1 are supplemented with the hormone. The regulatory characteristics of the P6 and P1 genes differ in several ways from those of the LSP genes. Expression of the P6 and P1 genes begins later than the LSP genes, and the levels of the transcripts remain high at the end of the third instar after the LSP transcripts have markedly decreased. Ecdysterone increases the level of the P1 transcript, but not of the P6 transcript, in ecdysterone-deficient ecd1 larvae.

Adipose Tissue↗