From owner-7tms_r@net.bio.net Wed Sep 04 23:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.erols.net!agate!newsgate.duke.edu!news.duq.edu!newsfeed.pitt.edu!newsflash.concordia.ca!sunqbc.risq.net!calvin.risq.qc.ca!news.mcgill.ca!news From: SAM Newsgroups: bionet.molbio.proteins.7tms_r Subject: gel shift Date: Thu, 05 Sep 1996 14:10:32 +0600 Organization: McGill University Lines: 5 Message-ID: <322E8AF8.29E2@musicb.mcgill.ca> NNTP-Posting-Host: embryo.pharma.mcgill.ca Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0 (Win16; I) CC: Anna I'm having a problem with the competition assay in the gel shift i.e. the cold oligo does not block or even decrease the binding of the hot oligo when incubated with a liver protein extract. I'm wondering if anyone ran into the same problem and/or has any ideas on what could cause it. Thank you. From owner-7tms_r@net.bio.net Wed Sep 04 23:00:00 1996 Path: biosci!bms.com!watson_j From: watson_j@bms.com (John Watson) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Hek 293 cell and non-hormonal inducibility Date: 5 Sep 1996 14:02:12 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 39 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Date: Thu, 05 Sep 1996 12:27:11 -0500 From: Tracy Blevins Subject: Hek 293 cell and non-hormonal inducibility X-Sender: tblevins@gsbs3.gs.uth.tmc.edu To: watson_j@bms.com MIME-version: 1.0 I am posting the following msg for Tracy Blevins (tblevins@gsbs3.gs.uth.tmc.edu) The University of Texas Houston Health Science Center Graduate School of Biomedical Sciences (w)792-8362 (h) 995-8248 Please do not respond to me. -------------------------------------------------------------------------------- I have found two non-hormonal systems for inducing expression of proteins in mammalian cells, TetON/OFF and Lac Switch. Unfortunately, the Tet system has been shown to give high background and low inducibility in the HEK 293 cells we use in our lab (JBC vol. 270 No.23 pp. 14168-14174, 1995). I have a few questions: 1. Are there any other non-hormonal systems out there? 2. Has anyone gotten a decent response with HEK 293 cells in the Tet system? 3. Has anyone tried the Lac Switch technology (with HEK 293 or other mammalian cells) and can tell me if it worked for you? Thanks for any advice. -- Tracy Blevins tblevins@gsbs3.gs.uth.tmc.edu The University of Texas Houston Health Science Center Graduate School of Biomedical Sciences (w)792-8362 (h) 995-8248 -------------------------------------------------------------------------------- From owner-7tms_r@net.bio.net Wed Sep 04 23:00:00 1996 Path: biosci!bms.com!watson_j From: watson_j@bms.com (John Watson) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: gel shift Date: 5 Sep 1996 14:02:12 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 14 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net SAM wrote: >I'm having a problem with the competition assay in the gel shift i.e. >the cold oligo does not block or even decrease the binding of the hot >oligo when incubated with a liver protein extract. I'm wondering if >anyone ran into the same problem and/or has any ideas on what could >cause it. Thank you. Um, not to be a wiseguy, but nonspecific binding, maybe? Are you using a nonspecific competitor (like poly dI:dC) in your rxns? AJW From owner-7tms_r@net.bio.net Wed Sep 04 23:00:00 1996 Path: biosci!CCR.DSI.UANL.MX!pearl From: pearl@CCR.DSI.UANL.MX ("Dr. Paul R.Earl") Newsgroups: bionet.molbio.proteins.7tms_r Subject: BIOMX, ELECTRONIC JOURNAL Date: 5 Sep 1996 14:58:28 -0700 Organization: UANL Lines: 8 Sender: daemon@net.bio.net Distribution: world Message-ID: <322F69FE.F3@ccr.dsi.uanl.mx> NNTP-Posting-Host: net.bio.net Please see http://www.uanl.mx/biomx or enter Biomx in Yahoo search to get the HomePage. Thank you for your kind attention. Best Wishes, Dr Paul R Earl From owner-7tms_r@net.bio.net Thu Sep 05 23:00:00 1996 Path: biosci!agate!howland.erols.net!newsfeed.internetmci.com!cpk-news-hub1.bbnplanet.com!newsserver.jvnc.net!netnews.sbphrd.com!news From: tom Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: Hela cells and adenylycylase Date: Fri, 06 Sep 1996 11:26:39 -0700 Organization: SmithKline Beecham Lines: 17 Message-ID: <32306CDF.DF7@sbphrd.com> References: <504tr9$q9p@oac2.hsc.uth.tmc.edu> NNTP-Posting-Host: 139.136.125.65 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.01 (Win16; I) Hello, I am looking for information about the Hela cell line. Specifically: 1. Does it express adenylylcylase (any type)? 2. Does it express G proteins? 3. Does it express beta 2 adrenergic receptors? I would appreciate any information you might have on this topic. Thanks, Tracy Blevins Test -- The opinions expressed in this communication are my own, and do not necessarily reflect those of my employer. From owner-7tms_r@net.bio.net Thu Sep 05 23:00:00 1996 Path: biosci!agate!spool.mu.edu!uwm.edu!cs.utexas.edu!howland.erols.net!newsserver.jvnc.net!netnews.sbphrd.com!news From: tom Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: Hela cells and adenylycylase Date: Fri, 06 Sep 1996 11:25:47 -0700 Organization: SmithKline Beecham Lines: 4 Message-ID: <32306CAB.7047@sbphrd.com> References: <504tr9$q9p@oac2.hsc.uth.tmc.edu> NNTP-Posting-Host: 139.136.125.65 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.01 (Win16; I) test -- The opinions expressed in this communication are my own, and do not necessarily reflect those of my employer. From owner-7tms_r@net.bio.net Thu Sep 05 23:00:00 1996 Path: biosci!agate!howland.erols.net!newsfeed.internetmci.com!cpk-news-hub1.bbnplanet.com!newsserver.jvnc.net!netnews.sbphrd.com!news From: tom Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: Hela cells and adenylycylase Date: Fri, 06 Sep 1996 11:21:15 -0700 Organization: SmithKline Beecham Lines: 30 Message-ID: <32306B9B.3EF5@sbphrd.com> References: <504tr9$q9p@oac2.hsc.uth.tmc.edu> NNTP-Posting-Host: 139.136.125.65 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.01 (Win16; I) Tracy Blevins wrote: > > Hello, > > I am looking for information about the Hela cell line. > Specifically: > 1. Does it express adenylylcylase (any type)? > 2. Does it express G proteins? > 3. Does it express beta 2 adrenergic receptors? > > I would appreciate any information you might have on this topic. > > Thanks, Tracy Blevins Sorry this is a test please ignore aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa bbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb ccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccc dddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddd eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee fffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffff ggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggg hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhh iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii -- The opinions expressed in this communication are my own, and do not necessarily reflect those of my employer. From owner-7tms_r@net.bio.net Thu Sep 05 23:00:00 1996 Path: biosci!agate!spool.mu.edu!uwm.edu!cs.utexas.edu!howland.erols.net!cam-news-hub1.bbnplanet.com!cpk-news-feed2.bbnplanet.com!cpk-news-hub1.bbnplanet.com!newsserver.jvnc.net!netnews.sbphrd.com!news From: Public_Access_Machine Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: Hela cells and adenylycylase Date: Fri, 06 Sep 1996 11:38:10 -0700 Organization: SmithKline Beecham Lines: 24 Message-ID: <32306F92.2B18@sbphrd.com> References: <504tr9$q9p@oac2.hsc.uth.tmc.edu> NNTP-Posting-Host: had610.ha.uk.sbphrd.com Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.01 (Win16; I) Tracy Blevins wrote: > > Hello, > > I am looking for information about the Hela cell line. > Specifically: > 1. Does it express adenylylcylase (any type)? > 2. Does it express G proteins? > 3. Does it express beta 2 adrenergic receptors? > > I would appreciate any information you might have on this topic. > > Thanks, Tracy Blevins I have measured adenylyl cyclase activity in HeLa cells expressing a recombinant 7TMR that couples positivley to AC. So HeLa cell obviously express an AC, which type(s) I don't know, and at least Gs. I hope this is of some use. Tim Young :) -- The opinions expressed in this communication are my own, and do not necessarily reflect those of my employer. From owner-7tms_r@net.bio.net Thu Sep 05 23:00:00 1996 Path: biosci!faseb.org!cpk-news-feed1.bbnplanet.com!cpk-news-feed2.bbnplanet.com!cpk-news-hub1.bbnplanet.com!newsserver.jvnc.net!crick.bms.com!usenet From: John Watson Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: Santa Cruz antibodies Date: Fri, 06 Sep 1996 11:34:24 -0400 Organization: Bristol-Myers Squibb Company Lines: 41 Message-ID: <32304480.11FB@bms.com> References: <3225C390.7CAB@pharm.emory.edu> Reply-To: watson_j@bms.com NNTP-Posting-Host: s107_static_223.wf.bms.com Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.0 (Macintosh; I; PPC) To: KPM KPM wrote: > > In response to recent postings about Santa Cruz antibodies, I wonder > if people would post their success, or lack of success, with these > reagents. > > I have heard from a number of people who have had no success with > the antibodies they obtained. Maybe an informal poll would be useful > in this forum. I have just finished testing by Western blot Santa Cruz antibodies directed against G(alpha)o, G(alpha)i, and G(alpha)i3 with the following results: Antibody Santa Cruz Claims My Observations -------- ----------------- --------------- Go (K-20) Specific for Go; no Cross-rxn with cross-rxn to other Gi3 alphas Gi (I-20) Highest affinity for Weak but detectable; Gi1; cross-rxn with Gi1=Gi3; Gi2 not detected Gi2 and Gi3 Gi3 (C-10) Reacts with all Gi Very strong for Gi3; but not other G alphas. Gi1 > Gi2 = Go. Westerns were performed against 200 ng purified G-protein alpha subunits obtained from Santa Cruz. All antisera were diluted 1:500. Immune complexes were visualized by enhanced chemiluminesence (Amersham ECL kit). AJW ---------------- John Watson Bristol-Myers Squibb Co. watson_j@bms.com --------------------------------------------------------------------- "If you're not part of the solution, you're part of the precipitate." --------------------------------------------------------------------- From owner-7tms_r@net.bio.net Thu Sep 05 23:00:00 1996 Path: biosci!bms.com!watson_j From: watson_j@bms.com (John Watson) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: Santa Cruz antibodies (REPOST) Date: 6 Sep 1996 10:57:39 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 47 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net I just looked at the formatting of my previous post. Let's try that again! --------------------------------------------------------------------------- KPM wrote: > > In response to recent postings about Santa Cruz antibodies, I wonder > if people would post their success, or lack of success, with these > reagents. > > I have heard from a number of people who have had no success with > the antibodies they obtained. Maybe an informal poll would be useful > in this forum. I have just finished testing by Western blot Santa Cruz antibodies directed against G(alpha)o, G(alpha)i, and G(alpha)i3 with the following results: Antibody Santa Cruz Claims My Observations -------- ----------------- --------------- Go (K-20) Specific for Go; no Cross-rxn with cross-rxn to other Gi3 alphas Gi (I-20) Highest affinity for Weak but detectable; Gi1; cross-rxn with Gi1=Gi3; Gi2 not detected Gi2 and Gi3 Gi3 (C-10) Reacts with all Gi Very strong for Gi3; but not other G Gi1 > Gi2 = Go. alphas. Westerns were performed against 200 ng purified G-protein alpha subunits obtained from Santa Cruz. All antisera were diluted 1:500. Immune complexes were visualized by enhanced chemiluminesence (Amersham ECL kit). AJW ---------------- John Watson Bristol-Myers Squibb Co. watson_j@bms.com --------------------------------------------------------------------- "If you're not part of the solution, you're part of the precipitate." --------------------------------------------------------------------- From owner-7tms_r@net.bio.net Fri Sep 06 23:00:00 1996 Path: biosci!IX.NETCOM.COM!jeffherz From: jeffherz@IX.NETCOM.COM (Jeffrey Herz, Ph.D.) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: affinity tags for receptor purification Date: 7 Sep 1996 10:49:35 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 26 Sender: daemon@net.bio.net Distribution: world Message-ID: <199609071748.KAA26939@dfw-ix2.ix.netcom.com> NNTP-Posting-Host: net.bio.net Hello, I would appreciate receiving any information or references regarding the use (worked examples and problems encountered) of affinity tags (eg. polyHis, FLAG, etc.) engineered into recombinant GPCR sequences and their use in receptor purification. Specifically: 1. Are there examples of both N- and C-terminal placements of the affinity tag? 2. Does the presence of the affinity tag alter the pharmacology of ligand binding? 2. Does the presence of the affinity tag have effects on receptor coupling or the ability to convert between high and low affinity states? Thank you in advance for any information on this topic. Sincerely, Jeffrey Herz From owner-7tms_r@net.bio.net Sun Sep 08 23:00:00 1996 Path: biosci!M.U-TOKYO.AC.JP!haga From: haga@M.U-TOKYO.AC.JP (Tatsuya Haga) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: affinity tags for receptor purification Date: 9 Sep 1996 01:13:12 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 49 Sender: daemon@net.bio.net Distribution: world Message-ID: <199609090805.RAA27775@mdpcws1.m.u-tokyo.ac.jp> NNTP-Posting-Host: net.bio.net >To: 7tms_r@net.bio.net >From: jeffherz@IX.NETCOM.COM (Jeffrey Herz, Ph.D.) >Subject: Re: affinity tags for receptor purification >Date: 7 Sep 1996 10:49:35 -0700 >Sender: daemon@net.bio.net >NNTP-Posting-Host: net.bio.net > >Hello, > >I would appreciate receiving any information or references regarding >the use (worked examples and problems encountered) of affinity tags >(eg. polyHis, FLAG, etc.) engineered into recombinant GPCR sequences >and their use in receptor purification. > >Specifically: > 1. Are there examples of both N- and C-terminal placements of the >affinity tag? > 2. Does the presence of the affinity tag alter the pharmacology of >ligand binding? > 2. Does the presence of the affinity tag have effects on receptor >coupling or the ability to convert between high and low affinity >states? > > >Thank you in advance for any information on this topic. > >Sincerely, > >Jeffrey Herz > > > > > > Dear Dr. Herz Rhodopsin and beta adrenergic receptors with poly histidine tag in their carboxyl terminus have been purified by using Ni-column and were shown to have essentially the same spectral and ligand binding properties as the wild types (Janssen et al., J. Biol. Chem. 270,11222-11229,1995; Kobilka, Anal. Biochem. 231`, 269-271, 1995). We have recently found that human m2 muscarinic acetylcholine receptors with poly histidine tag in their carboxyl terminus, which were expressed in SF9 cells, can be purified by using Co-column and can interact with and activate G protein Gi2 in the same way as the wild type receptor. The paper is under revision, and we hope that it will be published soon. Tatsuya Haga Tatsuya Haga From owner-7tms_r@net.bio.net Mon Sep 09 23:00:00 1996 Path: biosci!hubrecht.nl!markv From: markv@hubrecht.nl Newsgroups: bionet.molbio.proteins.7tms_r Subject: Gp expression plasmids Date: 10 Sep 1996 03:12:30 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 11 Sender: daemon@net.bio.net Distribution: world Message-ID: <199609101011.DAA03082@net.bio.net> NNTP-Posting-Host: net.bio.net Hi there, I am desperately seeking for plasmids expressing G-proteins in eukaryotic cells. Especially, the alfa-subunit of Gq and Gq11. Is there anyone out there who can help me? Thanks, Mark Verheijen Hubrecht Laboratory, Utrecht, The Netherlands, markv@hubrecht.nl From owner-7tms_r@net.bio.net Tue Sep 10 23:00:00 1996 Path: biosci!agate!spool.mu.edu!news.sgi.com!enews.sgi.com!www.nntp.primenet.com!nntp.primenet.com!howland.erols.net!newsfeed.internetmci.com!in1.uu.net!demos!news.glas.apc.org!glas!yur77 From: Dmitry Yuryev Newsgroups: bionet.molbio.proteins.7tms_r Subject: Receptor Subtypes Identification - Message-ID: Date: Wed, 11 Sep 1996 18:16:09 +0400 (WSU DST) X-Gateway: notes@glas.apc.org Lines: 38 CROSS-TITRATION: A new type of ligand-binding experiment for receptor subtypes identification > A new type of ligand-binding experiment called cross-titration experiment is being proposed for characterization of tissues containing multiple receptor subtypes. It is an extension of traditional inhibition experiment employing two different inhibitors at once: binding of a constant amount of labeled ligand is measured in approximately 10x10 array of wells where concentration of one inhibitor is varying in rows and concentration of another one - in columns. ... i 1/4 ^ 0 0 0 n ! h ! i 1/2 ! 0 0 0 b ! 2 ! 1 ! 0 0 0 ... ---------> 1 1/2 1/4 inhibitor1 The program CROSAFIN recovers from such data a two-dimensional distribution of receptor subtypes on the coordinate plane with affinity constant to one inhibitor on X-axis and to the second inhibitor - on Y-axis. Routinely, this method ensures a problem-free dealing with tissues containing 4-5 receptor subtypes, though, of course, still much depends upon availability of inhibitors with appropriate affinity profiles. > This program is now available from: ftp://garbo.uwasa.fi/pc/science/crsaf_01.zip From owner-7tms_r@net.bio.net Tue Sep 10 23:00:00 1996 Path: biosci!faseb.org!cpk-news-feed1.bbnplanet.com!cpk-news-feed2.bbnplanet.com!cam-news-hub1.bbnplanet.com!www.nntp.primenet.com!nntp.primenet.com!howland.erols.net!news.sprintlink.net!news-peer.sprintlink.net!newsfeed.internetmci.com!demos!news.glas.apc.org!glas!yur77 From: Dmitry Yuryev Newsgroups: bionet.molbio.proteins.7tms_r Subject: Many Troubles in Affinity Measureme Message-ID: Date: Thu, 12 Sep 1996 08:26:42 +0400 (WSU DST) X-Gateway: notes@glas.apc.org Lines: 36 Note papers describing several widespread errors in traditional methods of affinity analysis. Their summaries are below. Full texts (format MS Word 6.0 for Win) are available from: ftp://garbo.uwasa.fi/pc/science/crsaf_02.zip --------------------- ABSURD TRIVIAL ERRORS IN SCATCHARD PLOT ANALYSIS Dmitriy K.Yuryev yur77@glas.apc.org http://www.glasnet.ru/~yur77 Several types of widespread errors in performing Scatchard plot analysis resulting in fatal distortions of calculated affinities are surveyed more or less systematically. It appears that graphical analysis of non-linear Scathard plot "by eye" is almost impossible; and, moreover, results calculated by computer programs for affinity analysis often require additional "interpretation". Some of reported errors also definitely imply that data fabrication is quite common practice in this field. ----- REFINING CHENG-PRUSOFF EQUATION A generalisation of the Cheng-Prusoff formula relating affinity constant and the concentration of inhibitor giving 50% inhibition has been derived for the case when concentrations of ligands are not in a great excess. It shows that methods ensuring precise solving of binding equations (including computer approaches and equations derived in the present work) can not be used if these "Cheng-Prusoff conditions" of ligands' excess are not observed. The trouble is that beyond these conditions analysis becomes unreliable: the error contained in estimations of necessary parameters appears abruptly multiplied in the calculated affinity constant From owner-7tms_r@net.bio.net Wed Sep 11 23:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.erols.net!newsfeed.internetmci.com!news.compuserve.com!news.production.compuserve.com!news From: DSF <76042.532@CompuServe.COM> Newsgroups: bionet.molbio.proteins,bionet.molbio.proteins.7tms_r,bionet.molbio.yeast Subject: C-terminal epitope tags Date: 12 Sep 1996 19:35:46 GMT Organization: Cadus Lines: 8 Message-ID: <519omi$8va$1@mhadf.production.compuserve.com> Xref: biosci bionet.molbio.proteins:8743 bionet.molbio.proteins.7tms_r:859 bionet.molbio.yeast:5788 Hi- I have Flag, Flu, myc3, and AU1 c-terminal epitope tags on a seven-transmembrane receptor that I've been expressing in yeast. Since I didn't have a +ve control for flu, myc and au1, our vector man created a mammalian vector that I transiently transfected into CHO cells that shows binding in the membrane (the transfection worked fine) but there are no specific bands on westerns. Any ideas? Any help would be appreciated. Lynette From owner-7tms_r@net.bio.net Wed Sep 11 23:00:00 1996 Path: biosci!faseb.org!cpk-news-feed1.bbnplanet.com!cpk-news-feed2.bbnplanet.com!cam-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!newsfeed.internetmci.com!in1.uu.net!newsxfer2.itd.umich.edu!news.itd.umich.edu!usenet From: Rick Neubig Newsgroups: bionet.molbio.proteins.7tms_r Subject: G protein Postdocs - University of Michigan Date: Thu, 12 Sep 1996 07:48:48 -0400 Organization: University of Michigan Lines: 23 Message-ID: <3237F8A0.29F5@umich.edu> Reply-To: RNeubig@umich.edu NNTP-Posting-Host: warbler.med.umich.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0b6Gold (Win95; I) CC: taussig@umich.edu Postdoctoral positions in G protein signaling at the University of Michigan. Two NIH supported research positions are available immediately in the laboratories of Ron Taussig and Rick Neubig in the departments of Biological Chemistry and Pharmacology at the University of Michigan. Research areas include structure-function studies of adenylyl cyclase and G protein a or bg subunits by use of yeast genetic methods, expression in insect cells, mammalian cell transfection, photolabelling and proteolytic mapping, and synthetic peptides. Candidates should have a recent PhD in an appropriate field and experience in molecular biology, protein biochemistry, and signal transduction methods. Candidates should provide a cover letter outlining career objectives, a CV, and names of three references. FAX inquiries to Dr. Neubig at (313) 763-4450 or see http://www-personal.umich.edu/~rneubig/postdoc.html for further information. The University of Michigan is an Affirmative Action/Equal Opportunity Employer. Applications from qualified women, minorities and/or disabled individuals are encouraged. From owner-7tms_r@net.bio.net Wed Sep 11 23:00:00 1996 Path: biosci!internet!biosci!not-for-mail From: biohelp (BIOSCI Administrator) Newsgroups: bionet.molbio.proteins.7tms_r Subject: BIOSCI/bionet miniFAQ & Fundraiser Date: 12 Sep 1996 02:00:40 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 239 Sender: daemon@net.bio.net Distribution: world Message-ID: <199609120900.CAA16845@net.bio.net> NNTP-Posting-Host: net.bio.net (LAST REVISION: 30-JUL-95) This BIOSCI "miniFAQ" is designed to answer the questions that come up the *most frequently*. The main BIOSCI FAQ (Frequently Asked Questions) is accessible on the World Wide Web at URL http://www.bio.net/. If you can not find an answer to your question in this or other documentation, the BIOSCI technical support staff answers e-mail queries sent to biosci-help@net.bio.net We can only answer questions about the use of the newsgroups and mailing lists. We unfortunately do not have the staff to do Internet information searches or answer scientific questions. Please post those to the appropriate BIOSCI/bionet newsgroups. Contents: -------- 0) BIOSCI NEEDS YOUR SUPPORT!! 1) Using the WWW to access the BIOSCI/bionet newsgroups. 2) What to do about "spams," i.e., junk mail, ads, etc. 3) Examples of subscribing and unsubscribing to the mailing lists. 4) The BIOSCI user address and research interest directory. 0) BIOSCI NEEDS YOUR SUPPORT!! ------------------------------ BIOSCI's government funding has been expended, and we are now operating solely from advertising revenue that we have raised from our Web site at http://www.bio.net/. We need just a few minutes of your time to help us serve you. You can do two important things which will take very little time for you individually and will immensely help us continue to help you. First, please use our WWW system at http://www.bio.net/ to access the archives. You can post or reply to messages via your Web browser as described in item #1 below. Your usage helps attract sponsors. If you contact any of our sponsors, please be sure to thank them for supporting BIOSCI. It is critical for them to get this feedback if they are to continue their sponsorship for the long term. Second, if you work for a company or organization that provides products or services of interest to the biology community, please pass this message on to your marketing or marketing communications department or other appropriate group. Please ask them to help support BIOSCI by sponsoring our Web site and explain the uses and benefits of the system to the biology community. If they are interested, they can then contact us for further information at our tech support address, biosci-help@net.bio.net. 1) Using the WWW to access the BIOSCI/bionet newsgroups. -------------------------------------------------------- As of 10 December 1995, all BIOSCI/bionet full newsgroups are accessible through the World Wide Web (WWW) at URL http://www.bio.net. One can read and reply publicly or privately to both recent postings and archived messages through one's Web browser if it is configured properly to send e-mail. Each newsgroup is equipped with its own WAIS index. The main BIOSCI home page also has access to the BIO-JOURNALS Table of Contents database WAIS index and the BIOSCI user address database described in another item further below. 2) What to do about "spams," i.e., junk mail, ads, etc. ------------------------------------------------------- BIOSCI is a set of parallel USENET newsgroups (the "bionet" groups), mailing lists, and a hypermail archive at URL http://www.bio.net/. The same postings are distributed on all media (except for a small number of mailing-list-only groups at net.bio.net). Unfortunately it is becoming a despicable practice on the Internet (by a few people out to make a fast buck) to do automated mass postings to thousands of newsgroups and mailing lists. These attempts to grab free advertising are refered to as "spams" in the usual, somewhat boneheaded, net terminology. USENET is more susceptible to this practice, and many spams originate on the USENET groups and then are passed on to the mailing lists. However, spammers also get lists of mailing addresses and hit these too, so neither medium is immune. What should you do personally if you get junk mail? --------------------------------------------------- Just delete it and move on without reading it further. Filing a protest is becoming increasingly useless because spammers are often disguising the addresses where the messages are sent from. Unless you really understand Internet mail systems, your attempt at protest by sending replies to the message will often end up being sent to the address of an innocent person that the spammer is victimizing. What can BIOSCI/bionet do to protect its newsgroups? ---------------------------------------------------- The only solution currently available is to moderate the newsgroup. If this newsgroup is already moderated, then you are in good shape. Moderation protects the USENET distribution from about 95% of the spams that are being sent to date and protects the mailing lists completely. Moderation means, however, that someone has to take the time to review each message before it goes out. We have set up software here that simply allows the moderator to forward to an address at net.bio.net messages that (s)he wishes to have distributed. This takes no more time than that needed to read the message and pass it on, say about 1 min. per message. Most newsgroups currently have a discussion leader who is responsible for their newsgroup. The discussions leaders and their e-mail addresses are listed in the BIOSCI Information Sheet which is available on the Web at http://www.bio.net/. If a newsgroup is being hit with too many junk postings, please contact the discussion leader for that group and see if there is interest in moderating the group. Please do not assume that by simply posting a complaint to the newsgroup itself, anyone on the BIOSCI staff will act on your complaint. With close to 100 newsgroups to run, the BIOSCI staff has to rely on the discussion leaders of each newsgroup to report problems directly to us at biosci-help@net.bio.net. We will moderate any of our newsgroups if the discussion leader tells us that the readership of the group wishes to do so and if a moderator is willing to do the work. For most BIOSCI/bionet groups, this entails only a few minutes of work each day. Moderating a newsgroup will resolve probably 95% of the junk postings on the USENET distribution. Unfortunately there are easy ways for determined spammers to override the moderation mechanism on USENET, but we can protect our e-mail subscribers from unwanted postings if the newsgroup is moderated. You can also access our newsgroups over the WWW at URL http://www.bio.net. While this Web interface will not stop spammers from trying to post to the groups, this will give you yet another way, besides using USENET news, to keep the junk out of your personal mail files. For those of you with local USENET news systems, the Web interface will also give you faster access to new newsgroups and recent postings. 3) Examples of subscribing and unsubscribing to the mailing lists. ------------------------------------------------------------------ PLEASE NOTE: The BIOSCI management does NOT act on subscription/unsubscription requests that are posted improperly to the newsgroups and mailing lists. People who do this only bother everyone on the lists to no avail. Please be sure to follow the proper procedures below. Gory details are in the BIOSCI Information sheets on the Web at http://www.bio.net. Below we give an example utilizing the METHODS-AND-REAGENTS list at both of our two BIOSCI sites: Users in the Americas and Pacific Rim countries who use the BIOSCI ------------------------------------------------------------------ node at computer net.bio.net: ---------------------------- A) Determine the "listname" which is the <=8 character mail address ^^^^^^^^^^^^^ for the group. These can be found in the BIOSCI Info. Sheet. For the METHODS-AND-REAGENTS group the mailing address is methods@net.bio.net. The listname is the portion of the address to the left of the @ sign, i.e., "methods". The listname is used with the "subscribe" and "unsubscribe" commands illustrated below. B) Mail all commands in the body of a mail message addressed to biosci-server@net.bio.net. Do NOT send commands to the newsgroup posting addresses! Leave the Subject: line blank, any text on it will be ignored. C) In the body of your message put one or more of the following commands with an "end" command on the last line, e.g., subscribe methods unsubscribe methods end Do NOT put your e-mail address or other text on these lines. The server only allows you to cancel your subscription if the address on your mail header matches the address on our mailing list. Please ask for help at biosci-help@net.bio.net if your address has changed, e.g., if you know you are on the list but the server tells you that you are not a member. Users in Europe, Africa, and Central Asia who use the BIOSCI node at -------------------------------------------------------------------- computer daresbury.ac.uk (also known as dl.ac.uk): ------------------------------------------------- To subscribe and unsubscribe to/from the BIOSCI lists, you need to specify the full USENET newsgroup name with "bionet-news." prepended. The USENET newsgroup names are listed in the BIOSCI Information sheet on the Web at http://www.bio.net/. For the METHODS-AND-REAGENTS list the USENET newsgroup name is bionet.molbio.methds-reagnts, thus the appropriate commands are sub bionet-news.bionet.molbio.methds-reagnts unsub bionet-news.bionet.molbio.methds-reagnts These commands are included in a message addressed to mxt@dl.ac.uk, NOT to the newsgroup mailing addresses. As usual, include the text in the body of the message as text on the Subject: line is ignored. To unsubscribe from all the lists at the UK node, use unsub bionet-news Please note that if the address in the list is different than the one in your mail message header, you will not be able to unsubscribe by this method. If you have problems, please mail biosci@daresbury.ac.uk. 4) The BIOSCI user address and research interest directory. ----------------------------------------------------------- Please take this opportunity to add your name, address, and research interest information to the BIOSCI User Address Database if you have not already done so. You can fill out the address form directly through our Web page at URL http://www.bio.net/adrform.html. The address database is reindexed nightly for WWW access (the URL is http://www.bio.net/). If you are not directly on the Internet but can reach it by e-mail, please use our waismail server to access the user directory. waismail use is described above. You can also request a user address form by e-mail from biosci-help@net.bio.net. Please check your database entry from time-to-time to see if your address information is still up-to-date. Because of our limited personnel resources, we ask that you resubmit a *complete* form to revise your entry; we only replace complete entries and do not have resources to edit old forms. Sincerely, Dave Kristofferson BIOSCI/bionet Manager biosci-help@net.bio.net From owner-7tms_r@net.bio.net Thu Sep 12 23:00:00 1996 Path: biosci!Merck.Com!dennis_underwood From: dennis_underwood@Merck.Com (Dennis Underwood) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Hek 293 cell and non-hormon Date: 13 Sep 1996 06:36:37 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 48 Sender: daemon@net.bio.net Distribution: world Message-ID: <199609131331.JAA25768@igw2> NNTP-Posting-Host: net.bio.net Mail*Link(r) SMTP Hek 293 cell and non-hormon Date: Thu, 05 Sep 1996 12:27:11 -0500 From: Tracy Blevins Subject: Hek 293 cell and non-hormonal inducibility X-Sender: tblevins@gsbs3.gs.uth.tmc.edu To: watson_j@bms.com MIME-version: 1.0 I am posting the following msg for Tracy Blevins (tblevins@gsbs3.gs.uth.tmc.edu) The University of Texas Houston Health Science Center Graduate School of Biomedical Sciences (w)792-8362 (h) 995-8248 Please do not respond to me. -------------------------------------------------------------------------------- I have found two non-hormonal systems for inducing expression of proteins in mammalian cells, TetON/OFF and Lac Switch. Unfortunately, the Tet system has been shown to give high background and low inducibility in the HEK 293 cells we use in our lab (JBC vol. 270 No.23 pp. 14168-14174, 1995). I have a few questions: 1. Are there any other non-hormonal systems out there? 2. Has anyone gotten a decent response with HEK 293 cells in the Tet system? 3. Has anyone tried the Lac Switch technology (with HEK 293 or other mammalian cells) and can tell me if it worked for you? Thanks for any advice. -- Tracy Blevins tblevins@gsbs3.gs.uth.tmc.edu The University of Texas Houston Health Science Center Graduate School of Biomedical Sciences (w)792-8362 (h) 995-8248 -------------------------------------------------------------------------------- From owner-7tms_r@net.bio.net Thu Sep 12 23:00:00 1996 Path: biosci!faseb.org!cpk-news-feed1.bbnplanet.com!cpk-news-feed2.bbnplanet.com!cpk-news-hub1.bbnplanet.com!www.nntp.primenet.com!nntp.primenet.com!howland.erols.net!math.ohio-state.edu!newsfeed.acns.nwu.edu!news.cc.uic.edu!usenet From: blackman@tigger.uic.edu (Samuel C. Blackman) Newsgroups: bionet.molbio.proteins,bionet.molbio.proteins.7tms_r,bionet.molbio.methds-reagnts Subject: Anyone have the EBDA software package (or other Scatchard Analysis package)? Date: Fri, 13 Sep 1996 21:30:01 GMT Organization: University of Illinois at Chicago Lines: 20 Message-ID: <51cjn6$1a8s@piglet.cc.uic.edu> Reply-To: blackman@tigger.uic.edu NNTP-Posting-Host: f009.cme.uic.edu X-Newsreader: Forte Free Agent 1.0.82 Xref: biosci bionet.molbio.proteins:8757 bionet.molbio.proteins.7tms_r:861 bionet.molbio.methds-reagnts:49146 I was wondering if there were any free- or shareware programs available for Scatchard analysis of radioligand binding data (such as EBDA, LIGAND, etc.)? If so, drop me a note at blackman@tigger.uic.edu. Thanks! -- Sam Blackman -- Dept. of Pharmacology -- Univ. of Illinois at Chicago College of Medicine Samuel C. Blackman ! InterNet : blackman@tigger.uic.edu MD/PhD Student (3/7) ! Disclaimer : I speak for me, not UIC! Univ. of Ill. at Chicago ! Quote : "Quandro potro io finir di stupire?" Dept. of Pharmacology ! Phone : 312/996-4983 (lab) Fax: 312/996-1225 Check out my web page! ! http://www.uic.edu/~blackman/homepage.html "Research is to see what everybody else has seen, and to think what nobody else has thought." - Albert Szent-Gyorgyi From owner-7tms_r@net.bio.net Fri Sep 13 23:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!in1.uu.net!netnews.worldnet.att.net!newsadm From: Patrick Dorning Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: Gp expression plasmids Date: 14 Sep 1996 19:16:40 GMT Organization: AT&T WorldNet Services Lines: 2 Message-ID: <51f0ao$7hu@mtinsc01-mgt.ops.worldnet.att.net> References: <199609101011.DAA03082@net.bio.net> NNTP-Posting-Host: 106.middletown-55.va.dial-access.att.net Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.22ATT (Windows; U; 16bit) To: mick no we don't understand From owner-7tms_r@net.bio.net Sun Sep 15 23:00:00 1996 Path: biosci!FRODO.MGH.HARVARD.EDU!Kaplan From: Kaplan@FRODO.MGH.HARVARD.EDU (Josh Kaplan) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Go coupled receptors Date: 16 Sep 1996 11:42:20 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 14 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Dear Netters, Does anyone know of a molecularly cloned (preferably cDNA) 7-TMR that is specifically coupled to Go? Thanks much, Josh Joshua M. Kaplan, Ph.D. Department of Molecular Biology Wellman 8 Massachusetts General Hospital 50 Blossom St. Boston, MA 02114 Tel: 617-726-5962 Fax: 617-726-6893 From owner-7tms_r@net.bio.net Sun Sep 15 23:00:00 1996 Path: biosci!FRODO.MGH.HARVARD.EDU!Kaplan From: Kaplan@FRODO.MGH.HARVARD.EDU (Josh Kaplan) Newsgroups: bionet.molbio.proteins.7tms_r Subject: (none) Date: 16 Sep 1996 13:09:59 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 14 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Dear Netters, Does anyone know of a molecularly cloned (preferably cDNA) 7-TMR that is specifically coupled to Go? Thanks much, Josh Joshua M. Kaplan, Ph.D. Department of Molecular Biology Wellman 8 Massachusetts General Hospital 50 Blossom St. Boston, MA 02114 Tel: 617-726-5962 Fax: 617-726-6893 From owner-7tms_r@net.bio.net Sun Sep 15 23:00:00 1996 Path: biosci!COURRIER.USHERB.CA!stankova From: stankova@COURRIER.USHERB.CA (Jana Stankova) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Galpha expression vectors Date: 16 Sep 1996 13:16:52 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 7 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Hi, could someone point me in the direction where I could beg, borrow or buy the cDNAs for G14, G16 and PLCbeta2? many thanks in advance for any hints, JS From owner-7tms_r@net.bio.net Mon Sep 16 23:00:00 1996 Path: biosci!news.ohsu.edu!NewsWatcher!user From: forte@ohsu.edu (Michael Forte) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: Galpha expression vectors Date: 17 Sep 1996 22:36:38 GMT Organization: Vollum Institute Lines: 17 Distribution: world Message-ID: References: NNTP-Posting-Host: 137.53.73.42 I would contact Mel Simon at CalTech. Mike Forte In article , stankova@COURRIER.USHERB.CA (Jana Stankova) wrote: > Hi, > could someone point me in the direction where I could beg, borrow or buy > the cDNAs for G14, G16 and PLCbeta2? > many thanks in advance for any hints, > JS -- Mike Forte forte@ohsu.edu or forte@mtwo.com From owner-7tms_r@net.bio.net Tue Sep 17 23:00:00 1996 Path: biosci!agate!spool.mu.edu!newspump.sol.net!www.nntp.primenet.com!nntp.primenet.com!news.sprintlink.net!news-peer.sprintlink.net!vixen.cso.uiuc.edu!newsfeed.internetmci.com!in1.uu.net!fu-berlin.de!nntp.zit.th-darmstadt.de!voskovec.radio.cz!rznews.rrze.uni-erlangen.de!uni-erlangen.de!winx03!wpxx02!not-for-mail From: krasel@wpxx02.toxi.uni-wuerzburg.de (Cornelius Krasel) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: 2-hybrid on 7 TMS receptors Date: 18 Sep 1996 19:14:32 GMT Organization: University of Wuerzburg, Germany Lines: 20 Distribution: world Message-ID: <51phmo$5mh@winx03.informatik.uni-wuerzburg.de> References: NNTP-Posting-Host: wpxx02.toxi.uni-wuerzburg.de X-Newsreader: TIN [UNIX 1.3 950824BETA PL0] Henrik Dohlman (henrik.dohlman@YALE.EDU) wrote: > I'm interested in whether anyone has had any luck (good or bad) using 2-hybrid > screens with a G coupled receptor as a bait? Mark von Zastrow is doing this with receptor loops. When he gave his talk here (about half a year ago), he could only tell us that they had gotten clones, but they weren't sequenced then. I don't know whether anybody has tried this with full-length receptor. I would expect plenty of artifacts since you have a lot of hydrophobic stuff exposed which will bind about anything. Just my $0.02, --Cornelius. -- /* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */ /* D-97078 Wuerzburg, Germany email: phak004@rzbox.uni-wuerzburg.de SP3 */ /* "Science is the game we play with God to find out what His rules are." */ From owner-7tms_r@net.bio.net Tue Sep 17 23:00:00 1996 Path: biosci!daresbury!not-for-mail From: leurs@chem.vu.nl (Rob Leurs) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Ph.D. position open Date: 18 Sep 1996 17:16:29 +0100 Lines: 48 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <51p78t$m6l@mserv1.dl.ac.uk> Original-To: 7tms_r@dl.ac.uk >At the Leiden/Amsterdam Center of Drug Research a Ph.D. position (AiO) is >vacant within the division of Medicinal Chemistry. The project deals with the >structure-function relationship of the histamine H1 receptor. This receptor >protein belongs to the large family of G protein-coupled receptors and is the >target for a series of anti-allergic therapeutics (H1 antagonists). In recent >years we have been involved in the cloning of the gene encoding the H1 >receptor and have had a strong interest in delineation of the agonist binding >site and the regulation of H1 receptor function by agonists. In the present >project we would like to extend our research to the therapeutically important >H1 antagonists. >> >>The Leiden/Amsterdam Center of Drug Research is a collaborative research >>institute of the department of Pharmacochemistry of the Vrije Universtiteit >>(Amsterdam) and Biopharmaceutical Sciences of Leiden University. In the >>division of Medicinal Chemistry various G protein coupled receptors >>(histamine, adenosine, serotonin, adrenergic) are investigated by an >>interdisciplinary approach. Computational chemistry, drug synthesis, >>molecular biology and molecular biology are used to investigate various >>aspects of these drug targets. >> >>The project will be carried out at the Vrije Universiteit in Amsterdam. >> >>The candidate should have a strong background in molecular pharmacology and >>molecular biology. Experience with cell culture and/or baculovirus technology >>would be an advantage. Information can be obtained from Dr. Rob Leurs. >> >>Please send your application letter, CV and two references before October 15. >> >>Rob Leurs, Ph.D. ****************************************************************************** Rob Leurs, Ph.D Leiden/Amsterdam Center for Drug Research Division of Medicinal Chemistry Department of Pharmacochemistry, Vrije Universiteit De Boelelaan 1083, 1081 HV Amsterdam the Netherlands fax: 31204447610 tel: 31204447579 leurs@chem.vu.nl From owner-7tms_r@net.bio.net Tue Sep 17 23:00:00 1996 Path: biosci!YALE.EDU!henrik.dohlman From: henrik.dohlman@YALE.EDU ("Henrik Dohlman") Newsgroups: bionet.molbio.proteins.7tms_r Subject: 2-hybrid on 7 TMS receptors Date: 18 Sep 1996 06:35:17 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 5 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net I'm interested in whether anyone has had any luck (good or bad) using 2-hybrid screens with a G coupled receptor as a bait? Henrik Dohlman From owner-7tms_r@net.bio.net Wed Sep 18 23:00:00 1996 Path: biosci!ibpc.fr!cathy From: cathy@ibpc.fr Newsgroups: bionet.molbio.proteins.7tms_r Subject: Subscribe Date: 19 Sep 1996 00:43:44 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 7 Sender: daemon@net.bio.net Distribution: world Message-ID: <9609190747.AA27552@rigel.galaxy.ibpc.fr> NNTP-Posting-Host: net.bio.net I would like to subscribe to the 7TMS network. Could you tell me how to do Thanks a lot Bye Cathy ETCHEBEST IBPC 13 rue P. M Curie 75005 PARIS, FRANCE From owner-7tms_r@net.bio.net Wed Sep 18 23:00:00 1996 Path: biosci!rutgers!news.sgi.com!news.msfc.nasa.gov!newsfeed.internetmci.com!newsxfer2.itd.umich.edu!news.itd.umich.edu!usenet From: Rick Neubig Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: Go coupled receptors Date: Thu, 19 Sep 1996 19:05:06 -0400 Organization: University of Michigan Lines: 25 Message-ID: <3241D1A2.21B4@umich.edu> References: Reply-To: RNeubig@umich.edu NNTP-Posting-Host: warbler.med.umich.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0b6Gold (Win95; I) CC: Kaplan@FRODO.MGH.HARVARD.EDU Josh Kaplan wrote: > > Dear Netters, > Does anyone know of a molecularly cloned (preferably cDNA) 7-TMR that is > specifically coupled to Go? Thanks much, > Josh Josh, What do you mean by specifically? If you mean that it couples only to Go and to no other G protein? If so I don't think such a beast exists. Virtually all of the 7tmr's which couple to Go also couple (at least in vitro) to Gi also. There are plenty of cloned 7tmr's which couple to Go including the alpha2 adrenergic which I work on as well as adenosine, 5HT, muscarinic (m2 and m4), somatostatin, opioid etc. etc. Rick > > Joshua M. Kaplan, Ph.D. > Department of Molecular Biology > Wellman 8 > Massachusetts General Hospital > 50 Blossom St. > Boston, MA 02114 > Tel: 617-726-5962 Fax: 617-726-6893 From owner-7tms_r@net.bio.net Thu Sep 19 23:00:00 1996 Path: biosci!rutgers!uwm.edu!nntp.primenet.com!news.sgi.com!news.msfc.nasa.gov!newsfeed.internetmci.com!in1.uu.net!fu-berlin.de!nntp.zit.th-darmstadt.de!voskovec.radio.cz!rznews.rrze.uni-erlangen.de!uni-erlangen.de!winx03!wpxx02!not-for-mail From: krasel@wpxx02.toxi.uni-wuerzburg.de (Cornelius Krasel) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: 2-hybrid on 7 TMS receptors Date: 20 Sep 1996 08:29:14 GMT Organization: University of Wuerzburg, Germany Lines: 22 Distribution: world Message-ID: <51tkkq$mup@winx03.informatik.uni-wuerzburg.de> References: <51phmo$5mh@winx03.informatik.uni-wuerzburg.de> NNTP-Posting-Host: wpxx02.toxi.uni-wuerzburg.de X-Newsreader: TIN [UNIX 1.3 950824BETA PL0] Cornelius Krasel (krasel@wpxx02.toxi.uni-wuerzburg.de) wrote: > Henrik Dohlman (henrik.dohlman@YALE.EDU) wrote: > > I'm interested in whether anyone has had any luck (good or bad) using > > 2-hybrid screens with a G coupled receptor as a bait? > > Mark von Zastrow is doing this with receptor loops. When he gave his talk > here (about half a year ago), he could only tell us that they had gotten > clones, but they weren't sequenced then. I hate following up my own postings, but I stumbled upon a poster abstract: U. Klein, M. T. Ramirez, B. Kobilka, M. von Zastrow (1996): Studying Protein-Protein Interaction Involved in the Internalization of the Adrenergic Receptors. Biol. Chem. (formerly Biol. Chem. Hoppe-Seyler) 377 (Suppl.), S64 --Cornelius. -- /* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */ /* D-97078 Wuerzburg, Germany email: phak004@rzbox.uni-wuerzburg.de SP3 */ /* "Science is the game we play with God to find out what His rules are." */ From owner-7tms_r@net.bio.net Thu Sep 19 23:00:00 1996 Path: biosci!PHARM.MED.UPENN.EDU!mandana From: mandana@PHARM.MED.UPENN.EDU (Mandana Shahrestanifar) Newsgroups: bionet.molbio.proteins.7tms_r Subject: (none) Date: 20 Sep 1996 10:46:45 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 2 Sender: daemon@net.bio.net Distribution: world Message-ID: <199609201746.NAA05435@noc4.dccs.upenn.edu> NNTP-Posting-Host: net.bio.net subscribe From owner-7tms_r@net.bio.net Sun Sep 22 23:00:00 1996 Path: biosci!PHARM.MED.UPENN.EDU!mandana From: mandana@PHARM.MED.UPENN.EDU (Mandana Shahrestanifar) Newsgroups: bionet.molbio.proteins.7tms_r Subject: subscribe as a new member Date: 23 Sep 1996 07:31:49 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 2 Sender: daemon@net.bio.net Distribution: world Message-ID: <199609231431.KAA05205@noc4.dccs.upenn.edu> NNTP-Posting-Host: net.bio.net I would like to subscribe to your net. From owner-7tms_r@net.bio.net Sun Sep 22 23:00:00 1996 Path: biosci!rutgers!uwm.edu!cs.utexas.edu!howland.erols.net!EU.net!enews.sgi.com!news-feed.inet.tele.dk!news.inet.tele.dk!voskovec.radio.cz!rznews.rrze.uni-erlangen.de!uni-erlangen.de!winx03!wpxx02!not-for-mail From: krasel@wpxx02.toxi.uni-wuerzburg.de (Cornelius Krasel) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: 2-hybrid on 7 TMS receptors Date: 23 Sep 1996 19:20:58 GMT Organization: University of Wuerzburg, Germany Lines: 42 Distribution: world Message-ID: <526nuq$aac@winx03.informatik.uni-wuerzburg.de> References: <51phmo$5mh@winx03.informatik.uni-wuerzburg.de> <51tkkq$mup@winx03.informatik.uni-wuerzburg.de> NNTP-Posting-Host: wpxx02.toxi.uni-wuerzburg.de X-Newsreader: TIN [UNIX 1.3 950824BETA PL0] Cornelius Krasel (krasel@wpxx02.toxi.uni-wuerzburg.de) wrote: > I hate following up my own postings, but I stumbled upon a poster abstract: > > U. Klein, M. T. Ramirez, B. Kobilka, M. von Zastrow (1996): > Studying Protein-Protein Interaction Involved in the Internalization > of the Adrenergic Receptors. > Biol. Chem. (formerly Biol. Chem. Hoppe-Seyler) 377 (Suppl.), S64 Here is the text of the abstract (requested by several people; I don't think de Gruyter is going to sue me for breach of copyright :-). All typos my own. The abstract was presented at a meeting in Frankfurt/Main (Germany) which took place from March 26th to 28th. We are trying to identify proteins that interact with adrenergic receptors and thereby mediate and regulate their endocytosis. Even through agonist-mediated endocytosis of adrenergic receptors is phenomenologically well characterized, little is known about the underlying molecular mechanism. We have studied the alpha-2a- and alpha-2b-adrenergic receptor subtypes, which are structurally very similar but have clear differences in their subcellular trafficking. The alpha-2b-adrenergic receptor undergoes agonist- induced internalization while the alpha-2a-subtype does not. Domains and sequence motifs that determine the endocytosis phenotype of the alpha-2a- and alpha-2b-adrenergic receptor subtypes are being used as probes (baits) for the identification of interacting proteins using a yeast two-hybrid system and a cDNA library from HEK-293 cells. Classical biochemical methods are used to confirm the interaction of the receptor domains with the full-length proteins that are identified in the two-hybrid screening. Biochemical methods are also being used to identify interacting proteins de novo. As probes for the in vitro biochemical studies the domains that were used as baits in the two-hybrid screening are subcloned and expressed as GST-fusion proteins, which are then purified and used as ligands in Western blotting and affinity chromatography. --Cornelius. -- /* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */ /* D-97078 Wuerzburg, Germany email: phak004@rzbox.uni-wuerzburg.de SP3 */ /* "Science is the game we play with God to find out what His rules are." */ From owner-7tms_r@net.bio.net Tue Sep 24 23:00:00 1996 Path: biosci!aol.com!RickDLMN From: RickDLMN@aol.com Newsgroups: bionet.molbio.proteins.7tms_r Subject: subscribe Date: 25 Sep 1996 17:38:07 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 2 Sender: daemon@net.bio.net Distribution: world Message-ID: <960925203719_293304054@emout06.mail.aol.com> NNTP-Posting-Host: net.bio.net Please enter a subscription for me. Thank you. From owner-7tms_r@net.bio.net Wed Sep 25 23:00:00 1996 Path: biosci!daresbury!not-for-mail From: "D.J.Brownlee" Newsgroups: bionet.molbio.proteins.7tms_r Subject: assay/Gprotein/refs/techniques (fwd) Date: 26 Sep 1996 17:22:48 +0100 Lines: 55 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <52eako$1pf@mserv1.dl.ac.uk> Original-To: 7tms_r@dl.ac.uk Forwarded message: From djab Thu Sep 26 17:01:44 1996 Subject: assay/Gprotein/refs/techniques To: 7tms-r@dl.ac.uk Date: Thu, 26 Sep 1996 17:01:44 +0100 (BST) From: "D.J.Brownlee" X-Mailer: ELM [version 2.4 PL23] Content-Type: text Content-Length: 1011 I was wondering could I have some advice on secondary messenger systems in particular with respect to the following:- cGMP cAMP PLC Also which types of neurotransmitters cause their response via which type of G-protein? Would I be better using assay kits for the secondary messengers or RIA? Leading on from this, would it be better to prepare ElISA kits or purchase them made up? Are there any good antibodies about? I would be also grateful for a few good references (books, papers or otherwise). I will be working on an invertebrate system. I look forward to some help. Many thanks David Brownlee *************************************** Dr David J Brownlee Department of Physiology & Pharmacology School of Biological Sciences Bassett Crescent East University of Southampton Southampton SO16 7PX England, U.K. Tel. +44-1703-594368 Fax. +44-1703-594319 e-mail: djab@soton.ac.uk d_brownlee@msn.com **************************************** From owner-7tms_r@net.bio.net Thu Sep 26 23:00:00 1996 Path: biosci!agate!howland.erols.net!newsfeed.internetmci.com!newsgate.compuserve.com!news.production.compuserve.com!news From: DSF <76042.532@CompuServe.COM> Newsgroups: bionet.molbio.proteins,bionet.molbio.proteins.7tms_r,bionet.molbio.yeast Subject: AU1 Epitope tag Date: 25 Sep 1996 16:24:43 GMT Organization: Cadus Lines: 4 Message-ID: <52bmcb$qqf$1@mhadf.production.compuserve.com> Xref: biosci bionet.molbio.proteins:8869 bionet.molbio.proteins.7tms_r:881 bionet.molbio.yeast:5880 Anyone out there have any experience with AU1 epitope tag? Anything from anecdotal to referenced info is very welcome. Thanks Lynette From owner-7tms_r@net.bio.net Sun Sep 29 23:00:00 1996 Path: biosci!daresbury!not-for-mail From: Freddy de Bree Newsgroups: bionet.molbio.proteins.7tms_r Subject: change of address Date: 30 Sep 1996 09:29:09 +0100 Lines: 19 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <52o0cl$i1g@mserv1.dl.ac.uk> Original-To: 7tms_r@dl.ac.uk Please change address into: F.deBree@bris.ac.uk Thanks! ______________________________________ Freddy M. De Bree, PhD Protein Structure Group Department of Chemistry University of York Heslington York YO1 5DD tel: (0)1904-432599 fax: (0)1904-410519 ______________________________________