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R Ashfield

Publications and source records attributed to R Ashfield.

11 recordsLinked to original sources
PubMed1998

Cloning of the promoters for the beta-cell ATP-sensitive K-channel subunits Kir6.2 and SUR1.

The beta-cell ATP-sensitive potassium channel (K-ATP channel), which regulates insulin secretion, is composed of two types of subunits: 1) a sulfonylurea receptor (SUR1) and 2) an inwardly rectifying potassium channel (Kir6.2). We have isolated clones containing 5'-flanking DNA for both genes by hybridization screening of a human genomic library. Sequencing of over one kilobase of each upstream region has revealed that the putative promoters are G + C rich, with no TATA box. Several E-boxes and potential Sp1 sites are present in both promoters, and the Kir6.2 upstream region contains an Alu repeat. Using a luciferase reporter gene in transient transfection assays, we demonstrate that the upstream DNA contains promoters that are active in the beta-cell lines HIT T15 and MIN6. The promoters are completely inactive in the fibroblast cell line COS7 but show some activity in HepG2 (liver) and HEK293 (epithelial) cell lines. Deletion analysis suggests that a short (173-base pair [bp]) fragment of SUR1 5'-flanking sequence is sufficient for maximal promoter activity. In contrast, over 900 bp of Kir6.2 5' sequence are required for similar high level expression, and deletion of the Alu repeat results in an increase in promoter activity.

ATP-Binding Cassette Transporters↗
PubMed1997

Properties of cloned ATP-sensitive K+ currents expressed in Xenopus oocytes.

1. We have studied the electrophysiological properties of cloned ATP-sensitive K+ channels (KATP channels) heterologously expressed in Xenopus oocytes. This channel comprises a sulphonylurea receptor subunit (SUR) and an inwardly rectifying K+ channel subunit (Kir). 2. Oocytes injected with SUR1 and either Kir6.2 or Kir6.1 exhibited large inwardly rectifying K+ currents when cytosolic ATP levels were lowered by the metabolic inhibitors azide or FCCP. No currents were observed in response to azide in oocytes injected with Kir6.2, Kir6.1 or SUR1 alone, indicating that both the sulphonylurea receptor (SUR1) and an inward rectifier (Kir6.1 or Kir6.2) are needed for functional channel activity. 3. The pharmacological properties of Kir6.2-SUR1 currents resembled those of native beta-cell ATP-sensitive K+ channel currents (KATP currents): the currents were > 90% blocked by tolbutamide (500 microM), meglitinide (10 microM) or glibenclamide (100 nM), and activated 1.8-fold by diazoxide (340 microM), 1.4-fold by pinacidil (1 mM) and unaffected by cromakalim (0.5 mM). 4. Macroscopic Kir6.2-SUR1 currents in inside-out patches were inhibited by ATP with a Ki of 28 microM. Kir6.1-SUR1 currents ran down within seconds of patch excision preventing analysis of ATP sensitivity. 5. No sensitivity to tolbutamide or metabolic inhibition was observed when SUR1 was coexpressed with either Kir1.1a or Kir2.1, suggesting that these proteins do not couple in Xenopus ocytes. 6. Our data demonstrate that the Xenopus oocyte constitutes a good expression system for cloned KATP channels and that expression may be assayed by azide-induced metabolic inhibition.

Adenosine Triphosphate↗
PubMed1996

Promiscuous coupling between the sulphonylurea receptor and inwardly rectifying potassium channels.

Sulphonylureas are a class of drugs widely used to treat non-insulin-dependent diabetes mellitus. These drugs act by binding to a sulphonylurea receptor (SUR) in the pancreatic beta-cell membrane which inhibits an ATP-sensitive potassium (K-ATP) channel and thereby stimulates insulin secretion. There has been much debate as to whether SUR and the K-ATP channel are the same or separate proteins, whether SUR confers ATP-sensitivity on an ATP-insensitive pore-forming subunit, and whether sulphonylureas can also modulate other types of K-channel. We show here that SUR itself does not possess intrinsic channel activity but that it endows sulphonylurea sensitivity on several types of inwardly-rectifying K-channels. It does not necessarily confer ATP-sensitivity on these channels.

ATP-Binding Cassette Transporters↗
PubMed1995

Cloning and functional expression of the cDNA encoding an inwardly-rectifying potassium channel expressed in pancreatic beta-cells and in the brain.

A cDNA clone encoding an inwardly-rectifying K-channel (BIR1) was isolated from insulinoma cells. The predicted amino acid sequence shares 72% identity with the cardiac ATP-sensitive K-channel rcKATP (KATP-1;[6]). The mRNA is expressed in the brain and insulinoma cells. Heterologous expression in Xenopus oocytes produced currents which were K(+)-selective, time-independent and showed inward rectification. The currents were blocked by external barium and caesium, but insensitive to tolbutamide and diazoxide. In inside-out patches, channel activity was not blocked by 1 mM internal ATP. The sequence homology with KATP-1 suggests that BIR1 is a subunit of a brain and beta-cell KATP channel. However, pharmacological differences and the lack of ATP-sensitivity, suggest that if, this is the case, heterologous subunits must exert strong modulatory influences on the native channel.

Amino Acid Sequence↗
PubMed1994

MAZ-dependent termination between closely spaced human complement genes.

The zinc finger protein MAZ, originally identified as a factor that binds to the c-myc P2 promoter, is associated with transcriptional termination. As shown in these studies, a termination sequence between the closely spaced human complement genes C2 and Factor B contains a protein binding site which interacts with three different proteins in vitro. Binding of one of these factors, MAZ, correlates with activity of the C2 termination sequence in vivo. Cloned MAZ was used to obtain a consensus binding site, G5AG5. This allowed identification of new sites, between the closely spaced human genes g11 and C4 and within an intron of the mouse IgM-D gene, where termination is known to occur and regulate the expression of IgD. The g11 and IgM MAZ sites lie within sequences that have activity in a termination assay and, furthermore, mutation of C2 or g11 MAZ sites severely reduces termination activity. MAZ bends DNA, and inherently bent DNA is highly active as a terminator, suggesting that MAZ-induced bending is important for C2 and g11 termination. We propose that MAZ sites exist in promoters which require protection against transcriptional interference, such as those of closely spaced genes, to cause efficient termination. The MAZ consensus sequence will facilitate the identification of further sites.

Base Sequence↗
PubMed1991

Transcriptional termination between the closely linked human complement genes C2 and factor B: common termination factor for C2 and c-myc?

We have demonstrated, using a combination of nuclear run-off and poly(A) site competition assays, that transcriptional termination occurs between the closely spaced human complement genes, C2 and Factor B, soon after the C2 poly(A) site. A comparison of the C2 termination signal with a functionally similar sequence downstream of the human alpha 2 globin gene reveals that both signals function in an orientation dependent manner, with subfragments of the whole signal displaying partial effects. In the case of the C2 termination sequence a protein binds within it, and is partially responsible for the termination effect. We further demonstrate that the same (or closely related) protein binds to the ME1a1 site in the murine c-myc promoter, which has been implicated in c-myc attenuation. We suggest that the termination/pause sequences positioned downstream of a gene's poly(A) site may constitute the general signals that elicit transcriptional termination in genes transcribed by RNA polymerase II.

Base Sequence↗
PubMed1989

Multiple cooperative interactions constrain BPV-1 E2 dependent activation of transcription.

Transcription directed by the BPV-1 long control region (LCR) is conditional upon activation by the virally encoded E2 protein. Within the 1.0 kb LCR there are five separate regions, A to E, that contain E2 responsive enhancers. The smallest functional region, A, is only 38 bp and contains two copies of the consensus sequence ACC(N)6GGT that is known to function as an E2 binding site in vitro. We show that a pair of these constitutes a minimal functional E2 responsive enhancer element but that the strength of enhancer activity is dramatically reduced both by increasing the spacing between them and by removing the dual elements from the proximity of other key promoter elements. Furthermore, pairs of dual elements activated transcription to varying levels depending upon their spatial arrangement and promoter proximity. We have also identified a low level constitutive enhancer in the D region which lacks an E2 consensus binding site but which can be activated by E2. We show that the activation potential of this constitutive enhancer is increased by association with a single E2 binding site suggesting some cooperation/interaction between viral and cellular enhancer proteins.

Base Sequence↗