| 1. |
- Björklund, Stefan, et al.
(författare)
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An S-phase specific release from a transcriptional block regulates the expression of mouse ribonucleotide reductase R2 subunit
- 1992
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Ingår i: EMBO Journal. - 0261-4189 .- 1460-2075. ; 11:13, s. 4953-4959
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Tidskriftsartikel (refereegranskat)abstract
- Ribonucleotide reductase (RR) activity in mammalian cells is closely linked to DNA synthesis. The RR enzyme is composed of two non-identical subunits, proteins R1 and R2. Both proteins are required for holoenzyme activity, which is regulated by S-phase specific de novo synthesis and breakdown of the R2 subunit. In quiescent cells stimulated to proliferate and in elutriated cell populations enriched in the various cell cycle phases the R2 protein levels are correlated to R2 mRNA levels that are low in G0/G1-phase cells but increase dramatically at the G1/S border. Using an R2 promoter-luciferase reporter gene construct we demonstrate an unexpected early activation of the R2 promoter as cells pass from quiescence to proliferation. However, due to a transcriptional block, this promoter activation only results in very short R2 transcripts until cells enter the S-phase, when full-length R2 transcripts start to appear. The position for the transcriptional block was localized to a nucleotide sequence approximately 87 bp downstream from the first exon/intron boundary by S1 nuclease mapping of R2 transcripts from modified in vitro nuclear run-on experiments. These results identify blocking of transcription as a mechanism to control cell cycle regulated gene expression.
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| 2. |
- Blume-Jensen, Peter, et al.
(författare)
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Activation of the human c-kit product by ligand-induced dimerization mediates circular actin reorganization and chemotaxis
- 1991
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Ingår i: EMBO Journal. - 0261-4189 .- 1460-2075. ; 10:13, s. 4121-4128
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Tidskriftsartikel (refereegranskat)abstract
- The proto-oncogene c-kit is allelic with the murine white spotting (W) locus and encodes a transmembrane protein tyrosine kinase that is structurally related to the receptors for platelet-derived growth factor (PDGF) and colony-stimulating factor-1 (CSF-1). Recently the ligand for the c-kit product, stem cell factor (SCF), was identified in both transmembrane and soluble forms. In order to examine the mechanism for receptor activation by SCF and biological properties of the activated c-kit product, we transfected the wild-type human c-kit cDNA into porcine aortic endothelial cells. We found that the receptor was down-regulated and transmitted a mitogenic signal in response to stimulation with soluble SCF. We also demonstrate that SCF induces dimerization of the c-kit product in intact cells, and that dimerization of the receptor is correlated with activation of its kinase. Activation of the c-kit product by SCF was found to induce circular actin reorganization indistinguishable from that mediated by the PDGF beta-receptor in response to PDGF-BB. Furthermore, soluble SCF was a potent chemotactic agent for cells expressing the c-kit product, a property which might be of importance during embryonic development.
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| 3. |
- Doucas, V., et al.
(författare)
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Unregulated expression of c-Jun or c-Fos proteins but not Jun D inhibits oestrogen receptor activity in human breast cancer derived cells
- 1991
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Ingår i: EMBO Journal. - : Wiley-Blackwell. - 0261-4189 .- 1460-2075. ; 10:8, s. 2237-2245
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Tidskriftsartikel (refereegranskat)abstract
- We present evidence that oestrogen receptor activity in human MCF-7 breast cancer cells is reduced by over-expression of c-Jun or c-Fos proteins and to a lesser extent by Jun B overexpression. In contrast, overexpression of Jun D protein does not affect the activity of the oestrogen receptor. A region of c-Jun found to be required for repression of oestrogen receptor activity is located outside the DNA binding domain and is not conserved among the three Jun proteins. Finally, we suggest that c-Jun and c-Fos act independently to inactivate the oestrogen receptor.
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| 4. |
- Forrest, D, et al.
(författare)
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Distinct functions for thyroid hormone receptors alpha and beta in brain development indicated by differential expression of receptor genes.
- 1991
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Ingår i: EMBO Journal. - 0261-4189 .- 1460-2075. ; 10:2, s. 269-75
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Tidskriftsartikel (refereegranskat)abstract
- Thyroid hormones are essential for correct brain development, and since vertebrates express two thyroid hormone receptor genes (TR alpha and beta), we investigated TR gene expression during chick brain ontogenesis. In situ hybridization analyses showed that TR alpha mRNA was widely expressed from early embryonic stages, whereas TR beta was sharply induced after embryonic day 19 (E19), coinciding with the known hormone-sensitive period. Differential expression of TR mRNAs was striking in the cerebellum: TR beta mRNA was induced in white matter and granule cells after the migratory phase, suggesting a main TR beta function in late, hormone-dependent glial and neuronal maturation. In contrast, TR alpha mRNA was expressed in the earlier proliferating and migrating granule cells, and in the more mature granular and Purkinje cell layers after hatching, indicating a role for TR alpha in both immature and mature neural cells. Surprisingly, both TR genes were expressed in early cerebellar outgrowth at E9, before known hormone requirements, with TR beta mRNA restricted to the ventricular epithelium of the metencephalon and TR alpha expressed in migrating cells and the early granular layer. The results implicate TRs with distinct functions in the early embryonic brain as well as in the late phase of hormone requirement.
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| 5. |
- Ibáñez, C F, et al.
(författare)
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Structure-function studies of nerve growth factor : functional importance of highly conserved amino acid residues.
- 1990
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Ingår i: EMBO Journal. - 0261-4189 .- 1460-2075. ; 9:5, s. 1477-83
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Tidskriftsartikel (refereegranskat)abstract
- Selected amino acid residues in chicken nerve growth factor (NGF) were replaced by site-directed mutagenesis. Mutated NGF sequences were transiently expressed in COS cells and the yield of NGF protein in conditioned medium was quantified by Western blotting. Binding of each mutant to NGF receptors on PC12 cells was evaluated in a competition assay. The biological activity was determined by measuring stimulation of neurite outgrowth from chick sympathetic ganglia. The residues homologous to the proposed receptor binding site of insulin (Ser18, Met19, Val21, Asp23) were substituted by Ala. Replacement of Ser18, Met19 and Asp23 did not affect NGF activity. Modification of Val21 notably reduced both receptor binding and biological activity, suggesting that this residue is important to retain a fully active NGF. The highly conserved Tyr51 and Arg99 were converted into Phe and Lys respectively, without changing the biological properties of the molecule. However, binding and biological activity were greatly impaired after the simultaneous replacement of both Arg99 and Arg102 by Gly. The three conserved Trp residues at positions 20, 75 and 98 were substituted by Phe. The Trp mutated proteins retained 15-60% of receptor binding and 40-80% of biological activity, indicating that the Trp residues are not essential for NGF activity. However, replacement of Trp20 significantly reduced the amount of NGF in the medium, suggesting that this residue may be important for protein stability.
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| 6. |
- Ikonen, E, et al.
(författare)
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Aspartylglucosaminuria : cDNA encoding human aspartylglucosaminidase and the missense mutation causing the disease
- 1991
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Ingår i: EMBO Journal. - 0261-4189 .- 1460-2075. ; 10:1, s. 51-58
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Tidskriftsartikel (refereegranskat)abstract
- We have isolated a 2.1 kb cDNA which encodes human aspartylglucosaminidase (AGA, E.C. 3.5.1.26). The activity of this lysosomal enzyme is deficient in aspartylglucosaminuria (AGU), a recessively inherited lysosomal accumulation disease resulting in severe mental retardation. The polypeptide chain deduced from the AGA cDNA consists of 346 amino acids, has two potential N-glycosylation sites and 11 cysteine residues. Transient expression of this cDNA in COS-1 cells resulted in increased expression of immunoprecipitable AGA protein. Direct sequencing of amplified AGA cDNA from an AGU patient revealed a G----C transition resulting in the substitution of cysteine 163 with serine. This mutation was subsequently found in all the 20 analyzed Finnish AGU patients, in the heterozygous form in all 53 carriers and in none of 67 control individuals, suggesting that it represents the major AGU causing mutation enriched in this isolated population. Since the mutation produces a change in the predicted flexibility of the AGA polypeptide chain and removes an intramolecular S-S bridge, it most probably explains the deficient enzyme activity found in cells and tissues of AGU patients.
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| 7. |
- Åström, Jonas, et al.
(författare)
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In vitro deadenylation of mammalian mRNA by a HeLa cell 3' exonuclease
- 1991
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Ingår i: EMBO Journal. - 0261-4189 .- 1460-2075. ; 10:10, s. 3067-3071
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Tidskriftsartikel (refereegranskat)abstract
- We have identified a 3' exonuclease in HeLa cell extracts which deadenylates mammalian mRNA and leaves the mRNA body intact after poly(A) removal. Only homopolymeric adenosine tails located at the 3' end were efficiently removed by the exonuclease. The poly(A) removing activity did not require any specific sequences in the mRNA body either for poly(A) removal or for accumulation of the deadenylated mRNA. We conclude that the poly(A) removing activity is a 3' exonuclease since (i) reaction intermediates gradually lose the poly(A) tail, (ii) degradation is prevented by the presence of a cordycepin residue at the 3' end and (iii) RNAs having internally located poly(A) stretches are poor substrates for degradation. The possible involvement of the poly(A) removing enzyme in regulating mRNA translation and stability is discussed.
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| 8. |
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| 9. |
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| 10. |
- Fåhraeus, R, et al.
(författare)
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Response to cAMP levels of the Epstein-Barr virus EBNA2-inducible LMP1 oncogene and EBNA2 inhibition of a PP1-like activity.
- 1994
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Ingår i: The EMBO journal. - 0261-4189. ; 13:24, s. 6041-51
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Tidskriftsartikel (refereegranskat)abstract
- The expression of the Epstein-Barr virus LMP1 oncogene is regulated by viral and non-viral factors in a tissue dependent fashion. The virus encoded transcription factor EBNA2 induces its expression in human B-cells. However, this induction also requires the contribution of cellular and/or other viral factors. In nasopharyngeal carcinoma cells and in cells from Hodgkin's lymphoma, LMP1 gene transcription is independent of viral products. Here we show that the effect of a factor binding to a cAMP responsive-like element (CRE) in the LMP1 gene transcription regulatory sequence (LRS) is essential for efficient promoter activity in the DG75 B-cell line and that elevation of cAMP levels in the cells induces LRS-derived CAT activity in a CRE dependent fashion. Incubation of two EBV-immortalized B-cell lines expressing endogenous EBNA2A with 8-Br cAMP increased the levels of the latency associated 66 kDa LMP1 within 2 h. Interestingly, LMP1 expression in DG75 cells conferred resistance to the inhibitory effect of 8-Br cAMP on cell proliferation. The protein phosphatase 1 and 2A (PP1 and PP2A, respectively) inhibitor okadaic acid also stimulated LRS-CAT activity in DG75 cells. EBNA2A from an EBV-immortalized B-cell line co-immunopurified with a PP1-like protein. An EBNA2A fragment spanning residues 324-436 fused to the GST protein specifically rescued a PP1/PP2A-like component from DG75 cell extracts. This GST-EBNA2A fusion product inhibited a PP1-like activity in nuclear extracts from these cells.
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