From owner-fluorescent@net.bio.net Tue Apr 01 23:00:00 1997 Path: biosci!webtv.net!su-news-feed4.bbnplanet.com!su-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!news.maxwell.syr.edu!news.apfel.de!news-fra1.dfn.de!news-koe1.dfn.de!news.ruhr-uni-bochum.de!news.uni-stuttgart.de!news.urz.uni-heidelberg.de!paroppc1.zmbh.uni-heidelberg.de!user From: dietzel@sun0.urz.uni-heidelberg.de (Steffen Dietzel) Newsgroups: bionet.molbio.proteins.fluorescent Subject: Re: GFP fixing Date: 2 Apr 1997 07:58:16 GMT Organization: University of Heidelberg Lines: 51 Message-ID: References: <5feglt$a1k$1@news.fas.harvard.edu> <333F4E9E.DE2@sprintmail.com> NNTP-Posting-Host: paroppc1.zmbh.uni-heidelberg.de I did not do any quantitative measurements and I should mention that I work with a very bright GFP-signal. When I tried it I could see the GFP-Signal very well after 5 min in Methanol. However, morphology of the specimen is a different thing... I think I have not tried Ethanol after I found out that buffered Formaldehyde works fine for the fluorescence signal and quite well for morphology Steffen In article <333F4E9E.DE2@sprintmail.com>, Tom Frey wrote: > Steffen Dietzel wrote: > > > > (Robert Means) wrote: > > > > Can anyone give me a pointer to a protocol for visualizing GFP > > > infixed tissue? Thanks in advance. > > > > > > Bob Means > > > > You can use about every fixation method you want to, as long as pH is more > > or less neutral. Acidic Acid does not work for example. (Buffered) > > formaldehyd is fine. > > > > Steffen Dietzel > > > > Steffen > > I have heard/read other investigators suggest that ethanol fixation > works very > poorly for GFP. They suggest that the protein is either destroyed or > not well > retained upon rehydration. Could you let us know if this agrees with > your > experience? > > Tom -- Steffen Dietzel Zentrum fuer molekulare Biologie Heidelberg (ZMBH) University of Heidelberg Im Neuenheimer Feld 282 69120 Heidelberg, Germany Phone: +49/6221/54-6836 or -6879. Fax: -5893 listservmail:dietzel@sun0.urz.uni-heidelberg.de personal mail: dietzel@wotan.iwr.uni-heidelberg.de From owner-fluorescent@net.bio.net Tue Apr 01 23:00:00 1997 Message-ID: <33431863.2DD7@ana.unibe.ch> Date: Wed, 02 Apr 1997 18:39:31 -0800 From: Valentin Djonov Reply-To: Djonov@ana.unibe.ch Organization: Anatomy_University of Berne X-Mailer: Mozilla 3.0 (Win16; I) MIME-Version: 1.0 Newsgroups: bionet.molbio.proteins.fluorescent Subject: transgenic mice Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit NNTP-Posting-Host: anaval.unibe.ch Lines: 4 Path: biosci!daresbury!uninett.no!sn.no!nntp.uio.no!news.apfel.de!fu-berlin.de!unlisys!blackbush.xlink.net!news-ge.switch.ch!swsbe6.switch.ch!news.unibe.ch!anaval.unibe.ch Hallo, I am looking for mammary gland tumor model system - for example RAS or MYC transgenic mice. Thanks for yuor answer. From owner-fluorescent@net.bio.net Wed Apr 02 23:00:00 1997 Path: biosci!rutgers.rutgers.edu!gatech!csulb.edu!hammer.uoregon.edu!news-xfer.netaxs.com!cpk-news-hub1.bbnplanet.com!su-news-hub1.bbnplanet.com!news.bbnplanet.com!news.pbi.net!newsfeed.dacom.co.kr!nntp.kreonet.re.kr!newsfeed.kreonet.re.kr!news.postech.ac.kr!usenet From: "E.S. Shin" Newsgroups: bionet.molbio.proteins.fluorescent Subject: Transgenci tobacco expressing GFP Date: 3 Apr 1997 08:32:53 GMT Organization: POSTECH Lines: 19 Message-ID: <33436BC4.872@postech.ac.kr> Reply-To: capcoon@postech.ac.kr NNTP-Posting-Host: pmg12.postech.ac.kr Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0Gold (Win95; I) Dear Bionetter, I have made some transgenic tobacco that GFP is trasnsformed. GFP construct was designed to express by CaMV 35s promoter. I want to know how to test GFP expression in planta. In Fluorescent microscopy test, I observed green fluorecence in leaf vein. But I want to see green fluorescence in planta. In addition, Are there any netters who succeed in making transgenic plant with GFP? I wish your good advice Have a Nice results and Progress in your research. E.S. Shin Lab. of Plant Molecular Genetics Pohang University of Science and Technology Korea From owner-fluorescent@net.bio.net Thu Apr 03 23:00:00 1997 Path: biosci!ICRF.ICNET.UK!s.geley From: s.geley@ICRF.ICNET.UK (Stephan Geley) Newsgroups: bionet.molbio.proteins.fluorescent Subject: GFP and immunofluorescence Date: 4 Apr 1997 15:33:35 -0800 Organization: ICRF Lines: 7 Sender: daemon@net.bio.net Distribution: world Message-ID: <3345906F.3541@icrf.icnet.uk> NNTP-Posting-Host: net.bio.net I'm looking for some advive how to fix cells for immunostaining without loosing a GFP signal. So far, I' ve tried either methanol, methanol/acetone, or paraformaldehyde/TX100. However, none of these methods worked and I always loose the very bright GFP signal that I obtain in living cells. Thanks for your help, Stephan From owner-fluorescent@net.bio.net Fri Apr 04 23:00:00 1997 Path: biosci!daresbury!not-for-mail From: "Arjun.G." Newsgroups: bionet.molbio.proteins.fluorescent Subject: secretable GFP Date: 5 Apr 1997 09:35:11 +0100 Lines: 11 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <5i52rv$c81@mserv1.dl.ac.uk> Original-To: fluorpro@dl.ac.uk Is a secretable GFP available? Also, are organelle specific GFP's available? Thanks, Arjun. National Center for Biological Sciences, Bangalore 560 012, India. arjung@ncbs.tifrbng.res.in From owner-fluorescent@net.bio.net Sun Apr 06 23:00:00 1997 Path: biosci!PUB.ZJPTA.NET.CN!lidb From: lidb@PUB.ZJPTA.NET.CN (Jianping) Newsgroups: bionet.molbio.proteins.fluorescent Subject: call paper for the 3rd international symposium on plant pathology and biotechnology Date: 7 Apr 1997 19:24:15 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 108 Sender: daemon@net.bio.net Distribution: world Message-ID: <3349AB3D.3E18@pub.zjpta.net.cn> NNTP-Posting-Host: net.bio.net 3RD HANGZHOU INTERNATIONAL SYMPOSIUM ON PLANT PATHOLOGY AND BIOTECHNOLOGY HANGZHOU, P.R.CHINA (First Announcement) November 2- November 7, 1997 The 3rd Hangzhou International Symposium on Plant Pathology and Biotechnology will be hold at the Institute of Biotechnology , Zhejiang Agricultural University (IBZAU) from November 2 - November 7, 1997, sponsored by the Chinese Plant Pathology Society. LOCATION The symposium will be held in Hangzhou, one of the most famous scenic cities in China. Situated beside beautiful and enchanting West Lake, it is a thriving center of tourism, learning, commerce and international congress. SCIENTIFIC PROGRAM The scientific program will include plenary lectures, symposia, and posters on the following subjects, several invited professor will present their papers on plenary section. Mechanisms of Disease Resistance Molecular and Physiological Studies of the Plant-Pathogen Interaction Molecular Aspects of Pathogenesis Structure and Expression of Virus Genomes Novel Approaches to Diagnosis and Detection of Pathogens and Diseases Plant Diseases and Biotechnology Transformation and Transgenic Plant LANGUAGE The official language of the symposium will be English REQUIREMENT OF PAPER Space is available for posters. Researchers with new results and data in the areas relevant to plant pathology and biotechnology are encouraged to submit abstracts for poster display to ensure the broadest possible coverage and exchange of recent advances. Please notice the deadline for the submission of the abstract is Aug. 15, 1997. One page abstract should be written in English, typed in single spaced using 10-12 characters per inch and side margined of 1 1/4 inches (3 cm) and printed in a size of A4 in duplicates. Please mail them with a copy of abstract as a Microsoft Word for Windows 95 or Windows 3.X or Macintosh 3.X file on a floppy diskette. Please be sure to indicate the authors and their mailing addresses and e-mail addresses. The poster board will be 30 X 40 inches (about 75 cm X 95 cm) for each one. The title should be printed with letters at least 1 inch (2.4 cm). The official invitation will be forwarded on request. FEE AND ACCOMMODATION CHARGES The 100 USD registration fee for foreigner and 500RMB for Chinese is required. The accommodation is approximately 100 USD/day. Graduate students pay half of the cost. SPECIAL SUPPORTS Special supports could be granted. If the paper with high respective, all or part of accommodation fee for limited authors might be exempted. SOCIAL AND CULTURAL ACTIVITIES There will be a symposium banquet and sightseeing tours around West Lake and other scenic spots around Hangzhou. LIAISON The honor chairman for the symposium is Professor Liu Yi, who is the chairman of the Chinese Society for Plant Pathology. The chairman of the symposium is Li Debao, who is the vice chairman of Chinese Society for Plant Pathology and the Professor and Director of IBZAU. For more information please contact: Dr. Xu Ping or Dr. Xu Jianping with the address of: Institute of Biotechnology , Zhejiang Agricultural University, Hangzhou, Zhejiang 310029, P.R.China. Fax: +86-571-696 1525 Telephone: +86-571- 697 1182, 697 1184 E-mail: lidb@pub.zjpta.net.cn The list of members of organizing committee will be announced in the next circulation. From owner-fluorescent@net.bio.net Sun Apr 06 23:00:00 1997 Path: biosci!ZSULINK.ZSU.EDU.CN!lsbrc07 From: lsbrc07@ZSULINK.ZSU.EDU.CN (lsbrc07) Newsgroups: bionet.molbio.proteins.fluorescent Subject: S65TGFP expression in plant cell Date: 7 Apr 1997 05:20:54 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 20 Sender: daemon@net.bio.net Distribution: world Message-ID: <3349B989.563F@zsu.edu.cn> Reply-To: lsbrc07@zsulink.zsu.edu.cn NNTP-Posting-Host: net.bio.net We constructed an expression vector in which the coding sequence for S65TGFP was placed under the control of the rice actin1 (Act1) promoter. It was transformed into rice callus cells by particle bombardment and many bright green fluorescent dots could be seen after 6-8 hours. A few bright dots even could be seen 10 days after transformation.(The result was published in "Biotechnology Techniques" Vol.11, No.2, p.0133,1997) Furthermore, we obtained the R0 plants and seeds that regenerated from the transformed callus. By southern blot, it is proved that they all carry the S65TGFP gene in their chromosome. But we can not detect the fluorescence in the transgenic R0 planta and the shoots germinated from R1 seeds. It seemed that the two results contradict each other. Although someone had observed the splicing of a cryptic intron in GFP mRNA which abolished GFP expression in transgenic Arabidopsis plants, we consider that it is not enough to explain this contradiction. I wish your good advice. Your sincerely Huang Yue From owner-fluorescent@net.bio.net Sun Apr 06 23:00:00 1997 Path: biosci!image1.com!george_m From: george_m@image1.com ("Dr. George McNamara") Newsgroups: bionet.molbio.proteins.fluorescent Subject: Re: Expression of GFP in Bacillus subtilis Date: 7 Apr 1997 12:59:10 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 40 Sender: daemon@net.bio.net Distribution: world Message-ID: <3.0.32.19970407160327.00693ee8@popmail.image1.com> NNTP-Posting-Host: net.bio.net Dear Dr. Xu, C.D. Webb et al (1997) Cell 88:667-674 and (1995) J. Bacteriol. 177:5906-5911. The 1997 paper is in the March 7 issue and uses the very slick GFP-LacI binding to tandem repeats of the Lac operator. See also Straight et al (1996) Current Biology 6:1599-1608 and Robinett et al (1996) J. Cell Biol. 135:1685-1700 for use in other cell types and applications. Enjoy, geo p.s. You could have found the Webb et al (1995) paper with a Medline title search of "green" and "subtilis". >Has anyone expression the GFP in Bacillus subtilis? I hope it could be >expressed in B. subtilis without any inducer and it will be stable for >wild B. subtilus strain. > >Jianping Xu >Associate Frofessor >Biotechnology Institute >Zhejiang Agricultural University >268# Kaixuan Road >Hangzhou 310029 >China >E-mail: xujp@hotmail.com > > George McNamara, Ph.D. Applications Scientist Universal Imaging Corporation 502 Brandywine Parkway West Chester, PA 19380 USA voice: 610-344-9410 ext 224 fax: 610-344-9515 internet: www.image1.com From owner-fluorescent@net.bio.net Sun Apr 06 23:00:00 1997 Path: biosci!ZSULINK.ZSU.EDU.CN!lsbrc07 From: lsbrc07@ZSULINK.ZSU.EDU.CN Newsgroups: bionet.molbio.proteins.fluorescent Subject: S65TGFP expression in plant cell Date: 7 Apr 1997 03:12:13 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 21 Sender: daemon@net.bio.net Distribution: world Message-ID: <9704071013.AA00855@zsulink.zsu.edu.cn> NNTP-Posting-Host: net.bio.net We constructed an expression vector in which the coding sequence for S65TGFP was placed under the control of the rice actin1 (Act1) promoter. It was transformed into rice callus cells by particle bombardment and many bright green fluorescent dots could be seen after 6-8 hours. A few bright dots even could be seen 10 days after transformation.(The result was published in "Biotechnology Techniques" Vol.11, No.2, p.0133,1997) Furthermore, we obtained the R0 plants and seeds that regenerated from the transformed callus. By southern blot, it is proved that they all carry the S65TGFP gene in their chromosome. But we can not detect the fluorescence in the transgenic R0 planta and the shoots germinated from R1 seeds. It seemed that the two results contradict each other. Although someone had observed the splicing of a cryptic intron in GFP mRNA which abolished GFP expression in transgenic Arabidopsis plants, we consider that it is not enough to explain this contradiction. I wish your good advice. From owner-fluorescent@net.bio.net Sun Apr 06 23:00:00 1997 Path: biosci!HOTMAIL.COM!xujp From: xujp@HOTMAIL.COM (Xu Jianping) Newsgroups: bionet.molbio.proteins.fluorescent Subject: Expression of GFP in Bacillus subtilis Date: 7 Apr 1997 05:35:40 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 12 Sender: daemon@net.bio.net Distribution: world Message-ID: <3348E94C.97@hotmail.com> NNTP-Posting-Host: net.bio.net Has anyone expression the GFP in Bacillus subtilis? I hope it could be expressed in B. subtilis without any inducer and it will be stable for wild B. subtilus strain. Jianping Xu Associate Frofessor Biotechnology Institute Zhejiang Agricultural University 268# Kaixuan Road Hangzhou 310029 China E-mail: xujp@hotmail.com From owner-fluorescent@net.bio.net Sun Apr 06 23:00:00 1997 Path: biosci!ZSULINK.ZSU.EDU.CN!lsbrc07 From: lsbrc07@ZSULINK.ZSU.EDU.CN (lsbrc07) Newsgroups: bionet.molbio.proteins.fluorescent Subject: S65TGFP expression in plant cell Date: 7 Apr 1997 07:10:22 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 17 Sender: daemon@net.bio.net Distribution: world Message-ID: <3349D323.EDC@zsu.edu.cn> Reply-To: lsbrc07@zsulink.zsu.edu.cn NNTP-Posting-Host: net.bio.net We constructed an expression vector in which the coding sequence for S65TGFP was placed under the control of the rice actin1 (Act1) promoter. It was transformed into rice callus cells by particle bombardment and many bright green fluorescent dots could be seen after 6-8 hours. A few bright dots even could be seen 10 days after transformation.(The result was published in "Biotechnology Techniques" Vol.11, No.2, p.0133,1997) Furthermore, we obtained the R0 plants and seeds that regenerated from the transformed callus. By southern blot, it is proved that they all carry the S65TGFP gene in their chromosome. But we can not detect the fluorescence in the transgenic R0 planta and the shoots germinated from R1 seeds. It seemed that the two results contradict each other. Although someone had observed the splicing of a cryptic intron in GFP mRNA which abolished GFP expression in transgenic Arabidopsis plants, we consider that it is not enough to explain this contradiction. I wish your good advice. From owner-fluorescent@net.bio.net Mon Apr 07 23:00:00 1997 Path: biosci!ihnp4.ucsd.edu!munnari.OZ.AU!news.ecn.uoknor.edu!feed1.news.erols.com!news.maxwell.syr.edu!news.apfel.de!news-fra1.dfn.de!news-ge.switch.ch!serra.unipi.it!newsserver.cilea.it!oracle.csi.unimi.it!usenet From: clsmteam@imiucca.csi.unimi.it (Paolo Castano) Newsgroups: bionet.molbio.proteins.fluorescent Subject: IMMUNOFLUORESCENCE COURSE Date: Tue, 08 Apr 1997 14:28:26 GMT Organization: Istitute of Human Anatomy Lines: 15 Message-ID: <334a5604.28481042@news.csi.unimi.it> NNTP-Posting-Host: modem12.csi.unimi.it X-Newsreader: Forte Free Agent 1.1/32.230 Advanced International Immunofluorescence Course Gargnano '97 (Italy) The Advanced International Immunofluorescence Course is a post-doctorate theoretical/practical course, with propedeutical lectures and practical stages on traditional and confocal immunofluorescence microscopy and image and ion analysis. The course will take place in Gargnano (Lake of Garda) from 7 to 10 October 1997. Further information and registration details will be found at the following Web address http://imiucca.csi.unimi.it/endomi/ACIF.html Thank you Paolo Castano From owner-fluorescent@net.bio.net Mon Apr 07 23:00:00 1997 Path: biosci!GAS.UUG.ARIZONA.EDU!askewd From: askewd@GAS.UUG.ARIZONA.EDU (David J Askew) Newsgroups: bionet.molbio.proteins.fluorescent Subject: pKEN vector map Date: 8 Apr 1997 08:39:23 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 9 Sender: daemon@net.bio.net Distribution: world Message-ID: References: <334a5604.28481042@news.csi.unimi.it> NNTP-Posting-Host: net.bio.net Hello, My colleague and I are looking for a good map of the plasmid pKEN, which we wish to transfer the GFP sequence from. Any refrences will be much appreciated. Thank you, Dave Askew, Univ. of Arizona From owner-fluorescent@net.bio.net Tue Apr 08 23:00:00 1997 Path: biosci!LYSATOR.LIU.SE!ngn From: ngn@LYSATOR.LIU.SE (Niklas Gustavsson) Newsgroups: bionet.molbio.proteins.fluorescent Subject: Re: pKEN vector map Date: 9 Apr 1997 05:43:45 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 35 Sender: daemon@net.bio.net Distribution: world Message-ID: <3.0.32.19970409144441.006b49f4@pop.lysator.liu.se> NNTP-Posting-Host: net.bio.net > >My colleague and I are looking for a good map of the plasmid pKEN, which= =20 >we wish to transfer the GFP sequence from. Any refrences will be much >appreciated. > >Thank you, > >Dave Askew, Univ. of Arizona > Hi!=20 Try this reference: Ezaz-Nikpay, Uchino, Lerner & Verdine. Protein science(1994) 3:132-138. I= f the article doesn't satisfy you you can get the email-adress to Prof. G= reg Verdine, who made pKEN, from me. Regards Niklas Gustavsson ------------------ Niklas Gustavsson ngn@lysator.liu.se Phone +046 (0)13-14 19 47 (home) +046 (0)13-22 20 56 (University) http://www.lysator.liu.se/~ngn Member of Link=F6ping Graduate School in Biomedical Research Assistant webmaster for www.edu.isy.liu.se/matnat Former member of the Board for the branch MatNat, left with a huge parash= ute Former member of 4-verkeriet and GudFadderiet From owner-fluorescent@net.bio.net Tue Apr 08 23:00:00 1997 Path: biosci!FMI.CH!ludin From: ludin@FMI.CH (Beat Ludin) Newsgroups: bionet.molbio.proteins.fluorescent Subject: Keep GFP out of the nucleus Date: 9 Apr 1997 23:14:02 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 13 Sender: daemon@net.bio.net Distribution: world Message-ID: <199704100611.IAA17872@fmi.ch> NNTP-Posting-Host: net.bio.net Has anybody concatemerized GFP (or used another trick) in order to keep it out of the nucleus? Did it work? blu ----------------------------------------------------- Dr. Beat Ludin, FMI, PO Box 2543, 4002 Basel, Switzerland Tel. +41 61 697 6697 / FAX +41 61 697 3976 Internet:ludin@fmi.ch / Compuserve:100102,1527 From owner-fluorescent@net.bio.net Tue Apr 08 23:00:00 1997 Path: biosci!FMI.CH!ludin From: ludin@FMI.CH (Beat Ludin) Newsgroups: bionet.molbio.proteins.fluorescent Subject: GFP/cytoskeleton web sites Date: 9 Apr 1997 23:14:04 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 17 Sender: daemon@net.bio.net Distribution: world Message-ID: <199704100611.IAA17875@fmi.ch> NNTP-Posting-Host: net.bio.net I'm in the process of submitting a review on the use of GFP in cytoskeletal research. It will contain a list of relevant web sites. If you want your site listed or you know a site that matches the subject, please let me know the address (by private e-mail, if you wish). Thanks for your help blu ----------------------------------------------------- Dr. Beat Ludin, FMI, PO Box 2543, 4002 Basel, Switzerland Tel. +41 61 697 6697 / FAX +41 61 697 3976 Internet:ludin@fmi.ch / Compuserve:100102,1527 From owner-fluorescent@net.bio.net Wed Apr 09 23:00:00 1997 Path: biosci!unsw.edu.au!C.Marquis From: C.Marquis@unsw.edu.au (Chris Marquis) Newsgroups: bionet.molbio.proteins.fluorescent Subject: gfp antibodies, elisa and purification Date: 10 Apr 1997 22:32:04 -0700 Organization: Biotechnology, The University of New South Wales Lines: 13 Sender: daemon@net.bio.net Distribution: world Message-ID: <199704110532.PAA21599@sam.comms.unsw.EDU.AU> NNTP-Posting-Host: net.bio.net dear all, does anyone have experience in the use of ELISAs to quantitate EGFP or GFP. also are there any cheap sources of antibodies ? secondly, has anyone tried to purify GFP or eGFP. any experiences would be appreciated thanks chris From owner-fluorescent@net.bio.net Wed Apr 09 23:00:00 1997 Path: biosci!ruf.uni-freiburg.de!kohlersi From: kohlersi@ruf.uni-freiburg.de (Simon Kohler) Newsgroups: bionet.molbio.proteins.fluorescent Subject: IR-spectroscopy on isolated GFP Date: 10 Apr 1997 05:51:28 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 24 Sender: daemon@net.bio.net Distribution: world Message-ID: <1.5.4.32.19970410125234.0067dd1c@sun2.ruf.uni-freiburg.de> NNTP-Posting-Host: net.bio.net Does anyone knows whether there had been issues about IR-measurements on the isolated GFP ? If yes, i'm looking for them and i also need the procedure on how to isolate the expressed GFP from the cells. Can anyone send me some info about the procedure and if there had been issues on IR-spectroscopy before ? Thanx in advance Simon Kohler University of Freiburg /Germany e-mail: kohlersi@ruf.uni-freiburg.de ----------------------------------------------------------------- Simon Kohler | Bifaenge 108 | kohlersi@ruf.uni-freiburg.de 79111 Freiburg | T: 0761/4762938 | ----------------------------------------------------------------- "There's nothing like a sudden shock to clarify one's thoughts." From owner-fluorescent@net.bio.net Wed Apr 09 23:00:00 1997 Path: biosci!UTKUX.UTCC.UTK.EDU!vonarnim From: vonarnim@UTKUX.UTCC.UTK.EDU Newsgroups: bionet.molbio.proteins.fluorescent Subject: GFP-dimer Date: 10 Apr 1997 10:53:38 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 23 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net With regard to Beat Ludin's question about the GFP-dimer and its possible nuclear exclusion: Yes, I have made a dimer of GFP, and it is indeed excluded from the nucleus, at least much more efficiently than the monomer. The dimer is not twice as bright as the monomer, though. The trimer was only weakly fluorescent. Our assay is a transient expression assay in onion epidermal cells. Regards, Albrecht Albrecht von Arnim Dept. of Botany The University of Tennessee HBB 437 Knoxville, TN 37996-1100 Phone: (423) 974-6206 From owner-fluorescent@net.bio.net Wed Apr 09 23:00:00 1997 Path: biosci!AG.ARIZONA.EDU!dgalbrai From: dgalbrai@AG.ARIZONA.EDU (David Galbraith) Newsgroups: bionet.molbio.proteins.fluorescent Subject: Re: Keep GFP out of the nucleus Date: 10 Apr 1997 11:24:19 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 23 Sender: daemon@net.bio.net Distribution: world Message-ID: <3.0.1.32.19970410112331.00982d64@ag.arizona.edu> NNTP-Posting-Host: net.bio.net We fused it to GUS to exclude it from the nucleus. See Plant Journal, 11(3), 573-586 (1997). David Galbraith At 11:14 PM 4/9/97 -0700, you wrote: >Has anybody concatemerized GFP (or used another trick) in order to keep >it out of the nucleus? Did it work? > >blu > > > > >----------------------------------------------------- >Dr. Beat Ludin, FMI, PO Box 2543, 4002 Basel, Switzerland >Tel. +41 61 697 6697 / FAX +41 61 697 3976 >Internet:ludin@fmi.ch / Compuserve:100102,1527 > > > From owner-fluorescent@net.bio.net Wed Apr 09 23:00:00 1997 Path: biosci!ccr.jussieu.fr!geraud From: geraud@ccr.jussieu.fr (=?iso-8859-1?Q?G=E9rard?= Geraud) Newsgroups: bionet.molbio.proteins.fluorescent Subject: Subscribe Date: 10 Apr 1997 03:37:34 -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 subscribe fluopro thanks XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX X X X X Gerard GERAUD X Institut Jacques Monod X X Dept. Imagerie X 2, place Jussieu -Tour 43 X X Tel : 33 01 44 27 47 56 X 75251 Paris cedex 05 Fr. X X Em : geraud@ccr.jussieu.fr X X X Fax : 33 01 44 27 81 70 X X XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX From owner-fluorescent@net.bio.net Wed Apr 09 23:00:00 1997 Path: biosci!fcs280s.ncifcrf.gov!cpk-news-feed1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!news.maxwell.syr.edu!news.apfel.de!news-fra1.dfn.de!news-ber1.dfn.de!fu-berlin.de!news.belwue.de!news.uni-freiburg.de!inn From: kohlersi@ruf.uni-freiburg.de (Simon Kohler) Newsgroups: bionet.molbio.proteins.fluorescent Subject: IR-spectroscopy on the isolated GFP Date: Thu, 10 Apr 1997 12:41:48 +0200 Organization: Rechenzentrum der Universitaet Freiburg, Germany Lines: 14 Message-ID: NNTP-Posting-Host: slip71.home.uni-freiburg.de X-Newsreader: MicroPlanet Gravity v1.01 (30 Day Trial) Does anyone knows whether there had been issues about IR-measurements on the isolated GFP ? If yes, i'm looking for them and i also need the procedure on how to isolate the expressed GFP from the cells. Can anyone send me some info about the procedure and if there had been issues on IR-spectroscopy before ? Thanx in advance Simon Kohler University of Freiburg /Germany e-mail: kohlersi@ruf.uni-freiburg.de From owner-fluorescent@net.bio.net Wed Apr 09 23:00:00 1997 Path: biosci!FMI.CH!ludin From: ludin@FMI.CH (Beat Ludin) Newsgroups: bionet.molbio.proteins.fluorescent Subject: Re: GFP/cytoskeleton web sites Date: 10 Apr 1997 07:29:18 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 30 Sender: daemon@net.bio.net Distribution: world Message-ID: <199704101426.QAA21679@fmi.ch> NNTP-Posting-Host: net.bio.net Tobias Baskin wrote: > I saw your note. Where will your review be published? In Trends in Cell Biology (hopefully). > I am >thinking about getting involved with GFP-cytoskeleton and am wondering what >has been done. I would like to visualize microtubules in plant cells with >GFP. Are you aware of successful GFP microtubule work in animal cells Yes, we're doing it ourselves daily. > (or yeast)? Janet Carminati and Tim Stearns at Stanford are the yeast/tubulin-GFP experts. Good luck with your work and keep in touch. Beat ----------------------------------------------------- Dr. Beat Ludin, FMI, PO Box 2543, 4002 Basel, Switzerland Tel. +41 61 697 6697 / FAX +41 61 697 3976 Internet:ludin@fmi.ch / Compuserve:100102,1527 From owner-fluorescent@net.bio.net Wed Apr 09 23:00:00 1997 Path: biosci!daresbury!not-for-mail From: Beat Ludin Newsgroups: bionet.molbio.proteins.fluorescent Subject: GFP/cytoskeleton web sites Date: 10 Apr 1997 07:58:25 +0100 Lines: 17 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <5ii32h$der@mserv1.dl.ac.uk> x-sender: ludin@fmisun.fmi.ch x-mailer: Claris Emailer 2.0 x49, February 10, 1997 Original-To: I'm in the process of submitting a review on the use of GFP in cytoskeletal research. It will contain a list of relevant web sites. If you want your site listed or you know a site that matches the subject, please let me know the address (by private e-mail, if you wish). Thanks for your help blu ----------------------------------------------------- Dr. Beat Ludin, FMI, PO Box 2543, 4002 Basel, Switzerland Tel. +41 61 697 6697 / FAX +41 61 697 3976 Internet:ludin@fmi.ch / Compuserve:100102,1527 From owner-fluorescent@net.bio.net Thu Apr 10 23:00:00 1997 Path: biosci!internet!biosci!not-for-mail From: biohelp (BIOSCI Administrator) Newsgroups: bionet.molbio.proteins.fluorescent Subject: BIOSCI/bionet miniFAQ & Fundraiser Date: 11 Apr 1997 02:00:13 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 239 Sender: daemon@net.bio.net Distribution: world Message-ID: <199704110900.CAA08657@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-fluorescent@net.bio.net Sun Apr 13 23:00:00 1997 Path: biosci!image1.com!george_m From: george_m@image1.com ("Dr. George McNamara") Newsgroups: bionet.molbio.proteins.fluorescent Subject: Re: GFP in rec. plant virus Date: 14 Apr 1997 07:09:03 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 49 Sender: daemon@net.bio.net Distribution: world Message-ID: <3.0.32.19970414101131.006a6234@popmail.image1.com> NNTP-Posting-Host: net.bio.net Dear Olivier, Your best options are: EGFP from Clontech, max fluorescence at about 405 nm, still bright at 490 nm. or mGFP/GFP5 from Haseloff. See: K.R. Siemering, R. Golbik, R. Sever, J. Haseloff (1996) Mutations that suppress the thermostability of green fluorescent protein. Current Biology 6(12):1653-1663. (see article for spectra). Good luck! geo >Hi all! > >We are intending to create a recombinant plant virus containing a reporter >gene in order to locate the progress of infection. Colleagues of us will >soon buy a fluorescence microscope that we'll be allowed to use, however >we'll more probably routinely use a hand-held UV lamp. > >Could anyone please help us in the choice of the optimal GFP version to use >(wt, EGFP, S35T, uv, other...). > >Thanks for your help. >Olivier Le Gall > Pathologie Vegetale, INRA Bordeaux, BP 81 : > 33883 Villenave d'Ornon Cedex, FRANCE -,o >Tel +33(0)556 843 205 Fax +33(0)556 843 222 ( ) > legall@bordeaux.inra.fr /----"-"--- >* * * * * * *-* *-*-*-*-*-*-*-*-*-*-*-*-----/ W > > > George McNamara, Ph.D. Applications Scientist Universal Imaging Corporation 502 Brandywine Parkway West Chester, PA 19380 USA voice: 610-344-9410 ext 224 fax: 610-344-9515 internet: www.image1.com From owner-fluorescent@net.bio.net Sun Apr 13 23:00:00 1997 Path: biosci!CROP.UOGUELPH.CA!ACARLSON From: ACARLSON@CROP.UOGUELPH.CA ("Alvar Carlson") Newsgroups: bionet.molbio.proteins.fluorescent Subject: Plant sGFP expression Date: 14 Apr 1997 07:18:51 -0700 Organization: Crop Science, The Univ. of Guelph Lines: 12 Sender: daemon@net.bio.net Distribution: world Message-ID: <6D477AF58A6@csnet.nw.uoguelph.ca> NNTP-Posting-Host: net.bio.net Hello I am attempting to use sGFP to visually select transgenic barley. I am concerned that the accumulation of the GFP in the nucleus will be detrimental to the development of transgenic plants. Has anyone observed reduced transformation efficiency in plants when using GFP or reduced fertility in any of the transgenic plants generated? Furthermore, has anyone found that modified GFP, to exclude it from the nucleus, increases the transformation efficiency? Alvar Carlson University of Guelph From owner-fluorescent@net.bio.net Sun Apr 13 23:00:00 1997 Path: biosci!BORDEAUX.INRA.FR!legall From: legall@BORDEAUX.INRA.FR (Olivier Le Gall) Newsgroups: bionet.molbio.proteins.fluorescent Subject: GFP in rec. plant virus Date: 14 Apr 1997 02:23:05 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 18 Sender: daemon@net.bio.net Distribution: world Message-ID: <1.5.4.16.19970414102552.34176d32@bordeaux.bordeaux.inra.fr> NNTP-Posting-Host: net.bio.net Hi all! We are intending to create a recombinant plant virus containing a reporter gene in order to locate the progress of infection. Colleagues of us will soon buy a fluorescence microscope that we'll be allowed to use, however we'll more probably routinely use a hand-held UV lamp. Could anyone please help us in the choice of the optimal GFP version to use (wt, EGFP, S35T, uv, other...). Thanks for your help. Olivier Le Gall Pathologie Vegetale, INRA Bordeaux, BP 81 : 33883 Villenave d'Ornon Cedex, FRANCE -,o Tel +33(0)556 843 205 Fax +33(0)556 843 222 ( ) legall@bordeaux.inra.fr /----"-"--- * * * * * * *-* *-*-*-*-*-*-*-*-*-*-*-*-----/ W From owner-fluorescent@net.bio.net Sun Apr 13 23:00:00 1997 Path: biosci!daresbury!uninett.no!news-feed.inet.tele.dk!cpk-news-hub1.bbnplanet.com!su-news-hub1.bbnplanet.com!news.bbnplanet.com!newsout1.alt.net!news1.alt.net!news.aros.net!news.cs.utah.edu!news.cc.utah.edu!not-for-mail From: J.Ruplinger@m.cc.utah.edu (Rooster) Newsgroups: bionet.molbio.proteins.fluorescent Subject: Spirulina as food Date: Sun, 13 Apr 1997 09:16:09 GMT Organization: Jared Ruplinger Lines: 16 Message-ID: <5isscr$19f$4@news.cc.utah.edu> NNTP-Posting-Host: ctsasync60.cc.utah.edu X-Newsreader: Forte Free Agent 1.0.82 Hello. I Have heard that spirulina / blue-green algae can be used as a sole source of nutrition or food for extended periods of time. Has anyone had any experience with this or know of a location where I can get factual information or research results on this subject? Any help would be greatly appreciated!! Please email me at J.Ruplinger@m.cc.utah.edu Thanks Much!! Jared Ruplinger From owner-fluorescent@net.bio.net Mon Apr 14 23:00:00 1997 Path: biosci!FMI.CH!ludin From: ludin@FMI.CH (Beat Ludin) Newsgroups: bionet.molbio.proteins.fluorescent Subject: Re: intron in transgenic mice Date: 15 Apr 1997 23:59:03 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 15 Sender: daemon@net.bio.net Distribution: world Message-ID: <199704160656.IAA27222@fmi.ch> NNTP-Posting-Host: net.bio.net SAINTIGNY Yannick 161011 wrote: >Hello, in a transgenic mice, is it absolutely necessary to insert a >intron in the plsamid construction withe EGFP ? AFIK, current thinking says that an intron is usually required for efficient nuclear export of any mRNA. You will get much lower expression levels without, but I don't think that it as absolutely necessary ----------------------------------------------------- Dr. Beat Ludin, FMI, PO Box 2543, 4002 Basel, Switzerland Tel. +41 61 697 6697 / FAX +41 61 697 3976 Internet:ludin@fmi.ch / Compuserve:100102,1527 From owner-fluorescent@net.bio.net Mon Apr 14 23:00:00 1997 Path: biosci!fcs280s.ncifcrf.gov!cpk-news-feed1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!cam-news-hub1.bbnplanet.com!news.bbnplanet.com!uunet!in2.uu.net!202.232.2.100!np1.iij.ad.jp!wnoc-tyo-news!news.nc.u-tokyo.ac.jp!news From: Munehide Kano Newsgroups: bionet.molbio.proteins.fluorescent Subject: GFP in NIH3T3 Date: Tue, 15 Apr 1997 20:29:16 +0900 Organization: Network Operation Centre, The University of Tokyo Lines: 8 Message-ID: <3353667A.53FC@m.u-tokyo.ac.jp> NNTP-Posting-Host: 130.69.111.126 Mime-Version: 1.0 Content-Type: text/plain; charset=iso-2022-jp Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0Gold (Macintosh; I; PPC) I am trying to express GFP in NIH3T3 cell line. But GFP fluorescence signal is very poor in NIH3T3. Otherwise, expression in Rat-2 cell line is very strong. Isn't NIH3T3 suitable cell line for GFP? Thanks for your knowredges about GFP in NIH3T3. Munehide Kano mkano@m.u-tokyo.ac.jp From owner-fluorescent@net.bio.net Mon Apr 14 23:00:00 1997 Path: biosci!FMI.CH!ludin From: ludin@FMI.CH (Beat Ludin) Newsgroups: bionet.molbio.proteins.fluorescent Subject: Re: GFP in NIH3T3 Date: 15 Apr 1997 23:55:35 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 19 Sender: daemon@net.bio.net Distribution: world Message-ID: <199704160652.IAA26635@fmi.ch> NNTP-Posting-Host: net.bio.net >>I am trying to express GFP in NIH3T3 cell line. But GFP fluorescence >>signal is very poor in NIH3T3. Otherwise, expression in Rat-2 cell line >>is very strong. >>Isn't NIH3T3 suitable cell line for GFP? > > I usually express GFP in NIH3T3 with a great fluorescence. > Same here. Sounds like a promotor issue. blu ----------------------------------------------------- Dr. Beat Ludin, FMI, PO Box 2543, 4002 Basel, Switzerland Tel. +41 61 697 6697 / FAX +41 61 697 3976 Internet:ludin@fmi.ch / Compuserve:100102,1527 From owner-fluorescent@net.bio.net Mon Apr 14 23:00:00 1997 Path: biosci!daresbury!not-for-mail From: SAINTIGNY Yannick 161011 Newsgroups: bionet.molbio.proteins.fluorescent Subject: intron in transgenic mice Date: 16 Apr 1997 07:15:14 +0100 Lines: 2 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <5j1qpi$bf@mserv1.dl.ac.uk> MIME-Version: 1.0 Original-To: "'Liste EGFP'" Hello, in a transgenic mice, is it absolutely necessary to insert a intron in the plsamid construction withe EGFP ? From owner-fluorescent@net.bio.net Mon Apr 14 23:00:00 1997 Path: biosci!daresbury!uninett.no!news-feed.inet.tele.dk!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!ais.net!uunet!in3.uu.net!202.232.2.100!np1.iij.ad.jp!wnoc-tyo-news!aist-nara!wnoc-kyo-news!kuis-news!anes5.kuhp.kyoto-u.ac.jp!user From: khirota@kuhp.kyoto-u.ac.jp (Kiichi Hirota) Newsgroups: bionet.molbio.proteins.fluorescent Subject: Re: GFP in NIH3T3 Date: Wed, 16 Apr 1997 13:42:18 +0900 Organization: Department of Anesthesia, KYOTO University Hospital Lines: 17 Message-ID: References: <3353667A.53FC@m.u-tokyo.ac.jp> NNTP-Posting-Host: anes5.kuhp.kyoto-u.ac.jp In article <3353667A.53FC@m.u-tokyo.ac.jp>, Munehide Kano wrote: >I am trying to express GFP in NIH3T3 cell line. But GFP fluorescence >signal is very poor in NIH3T3. Otherwise, expression in Rat-2 cell line >is very strong. >Isn't NIH3T3 suitable cell line for GFP? I usually express GFP in NIH3T3 with a great fluorescence. -- *************************************************** Kiichi Hirota, MD Department of Anesthesia, Kyoto University Hospital Sakyo-ku, Kyoto 606-01, Japan Phone: +81(Jpn)-75-751-3436 Fax: +81(Jpn)-75-752-3259 E-mail: khirota@kuhp.kyoto-u.ac.jp From owner-fluorescent@net.bio.net Mon Apr 14 23:00:00 1997 Path: biosci!daresbury!uninett.no!news-feed.inet.tele.dk!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!rill.news.pipex.net!pipex!uknet!usenet1.news.uk.psi.net!uknet!uknet!lyra.csx.cam.ac.uk!news From: Dr Mike Witty Newsgroups: bionet.molbio.proteins.fluorescent Subject: GFP fusion proteins Date: 15 Apr 1997 12:38:05 GMT Organization: Department of Plant Sciences, Cambridge Lines: 5 Message-ID: <5ivsrd$36g@lyra.csx.cam.ac.uk> NNTP-Posting-Host: molgen4.plantsci.cam.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; 68K) X-URL: news:bionet.molbio.proteins.fluorescent Does anyone know about GFP fusions and if they retain activity? The one example I know of if GFP fusion to protein A, touted as a good idea for Western blotting. Mike Witty. From owner-fluorescent@net.bio.net Tue Apr 15 23:00:00 1997 Path: biosci!AVIRON.COM!dstec From: dstec@AVIRON.COM (David Stec) Newsgroups: bionet.molbio.proteins.fluorescent Subject: (none) Date: 16 Apr 1997 12:17:51 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 3 Sender: daemon@net.bio.net Distribution: world Message-ID: References: <5j3186$nhd@mserv1.dl.ac.uk> NNTP-Posting-Host: net.bio.net unsubscribe David Stec From owner-fluorescent@net.bio.net Tue Apr 15 23:00:00 1997 Path: biosci!POPMAIL.UCSD.EDU!sfarlow From: sfarlow@POPMAIL.UCSD.EDU (sam farlow) Newsgroups: bionet.molbio.proteins.fluorescent Subject: (none) Date: 16 Apr 1997 13:07:42 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 2 Sender: daemon@net.bio.net Distribution: world Message-ID: <199704162008.NAA12633@popmail.UCSD.EDU> NNTP-Posting-Host: net.bio.net unsubscribe From owner-fluorescent@net.bio.net Tue Apr 15 23:00:00 1997 Path: biosci!rutgers.rutgers.edu!gatech!news-out.communique.net!communique!news-spur1.maxwell.syr.edu!news.maxwell.syr.edu!news-peer.sprintlink.net!news.sprintlink.net!sprint!newsfeed.internetmci.com!news-was.dfn.de!news-fra1.dfn.de!news-ge.switch.ch!feed2.belnet.be!news.belnet.be!alpha.luc.ac.be!pstn11.luc.ac.be!user From: ghermans@luc.ac.be (Guy Hermans) Newsgroups: bionet.molbio.proteins.fluorescent Subject: pTRACER stability without T antigen?? Date: Wed, 16 Apr 1997 17:52:04 +0100 Organization: Dr. L. Willems-Instituut Lines: 26 Message-ID: Reply-To: hellings@luc.ac.be NNTP-Posting-Host: pstn11.luc.ac.be I'm considering of using the new pTRACER vectors of Invitrogen. It are vectors with an SV40 ori and as tracer GFP. I'm planning of using them to express a gene in lymphocytes. My questions: 1. Is it possible to use this vector in transfections of T lymphocytes? They don't have the large T antigen, and I wondered if the T antigen is a necessary factor? It doesn't say anything in the folder I got. What will theoretically happen with the SV40 plasmid vector? Will it replicate together with the cells? It is possible to select for an antibiotic. 2. Does anyone have experience with this at all? Thanks for the help niels hellings -- Niels Hellings, PhD student Ms research Unit Immunology research group Dr. L. Willems-Institute Dept. of Physiology, LUC University Campus University Campus B-3590 Diepenbeek B-3590 Diepenbeek Belgium Belgium Voice ++32(0)11/26.92.07 Fax ++32(0)11/26.92.09 From owner-fluorescent@net.bio.net Tue Apr 15 23:00:00 1997 Path: biosci!daresbury!not-for-mail From: "Arjun.G." Newsgroups: bionet.molbio.proteins.fluorescent Subject: GPI anchored GFP Date: 16 Apr 1997 18:11:34 +0100 Lines: 7 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <5j3186$nhd@mserv1.dl.ac.uk> Original-To: fluorpro@dl.ac.uk Is anyone aware of a GPI-anchored GFP molecule? Have any attempts been made to create such a molecule? Arjun. National Center for Biological Sciences. Bangalore 560 012, India. From owner-fluorescent@net.bio.net Wed Apr 16 23:00:00 1997 Path: biosci!TC.UMN.EDU!Dean.J.Aguiar-1 From: Dean.J.Aguiar-1@TC.UMN.EDU (Dean J Aguiar) Newsgroups: bionet.molbio.proteins.fluorescent Subject: No Subject Date: 17 Apr 1997 12:24:29 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 2 Sender: daemon@net.bio.net Distribution: world Message-ID: <3356792473c1844@mhub2.tc.umn.edu> NNTP-Posting-Host: net.bio.net unsubscribe From owner-fluorescent@net.bio.net Wed Apr 16 23:00:00 1997 Newsgroups: bionet.molbio.proteins.fluorescent Path: biosci!agate!nntpfeed.doc.ic.ac.uk!sunsite.doc.ic.ac.uk!warwick!bris.ac.uk!usenet From: Andrew Doherty Subject: Re: GFP fusion proteins X-Nntp-Posting-Host: anaajd.ana.bris.ac.uk Content-Type: text/plain; charset=us-ascii To: Dr Mike Witty Message-ID: <3355E64C.56FF@Bris.ac.uk> Sender: usenet@fsa.bris.ac.uk (Usenet) Content-Transfer-Encoding: 7bit Organization: University of Bristol, UK References: <5ivsrd$36g@lyra.csx.cam.ac.uk> Mime-Version: 1.0 Date: Thu, 17 Apr 1997 08:58:52 GMT X-Mailer: Mozilla 3.0 (Win95; I) Lines: 33 Dr Mike Witty wrote: > > Does anyone know about GFP fusions and if they retain activity? The one > example I know of if GFP fusion to protein A, touted as a good idea for > Western blotting. Mike Witty. Yeah, a recent paper from Caron's lab used a B2-adrenergic receptor-GFP fusion to look at internalisation and showed that the native, epitope tagged receptor and the fusion (also epitope tagged) were functionally indistiguishable., The paper is: Barak L.S. et al (1997) Internal trafficking and suface mobility of a functionally intact B2-adrenergic receptor-green fluorescent protein conjugate. Mol. Pharm. 51; pp177-184 We're currently making AMPA receptor GFP fusions, and seem to get correct localisation when expressed in HEK293's, but we have no functional data as yet. Hope it helps Andy D -- ************************************************************* Dr Andrew Doherty email - a.doherty@bris.ac.uk Dept. Anatomy Tel (0117)9287421 School of Medical Sciences Fax (0117)9287402 University of Bristol University Walk Bristol UK BS8 1TD ************************************************************* From owner-fluorescent@net.bio.net Wed Apr 16 23:00:00 1997 Path: biosci!uoguelph.ca!jtrevors From: jtrevors@uoguelph.ca (Professor J. T. Trevors) Newsgroups: bionet.molbio.proteins.fluorescent Subject: unsubscribe Date: 17 Apr 1997 10:49:02 -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 unsubscribe Professor J. T. Trevors Department of Environmental Biology Rm 3220 Bovey Building University of Guelph Guelph, Ontario Canada N1G 2W1 Tel: 519-824-4120 ext. 3367 Fax: 519-837-0442 Email:jtrevors@uoguelph.ca From owner-fluorescent@net.bio.net Wed Apr 16 23:00:00 1997 Path: biosci!nki.nl!wubbo From: wubbo@nki.nl (Richard Wubbolts) Newsgroups: bionet.molbio.proteins.fluorescent Subject: unsubscribe Date: 17 Apr 1997 09:56:03 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 2 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net unsubscribe From owner-fluorescent@net.bio.net Wed Apr 16 23:00:00 1997 Path: biosci!bs.aist-nara.ac.jp!kimata From: kimata@bs.aist-nara.ac.jp (Yukio KIMATA) Newsgroups: bionet.molbio.proteins.fluorescent Subject: (none) Date: 17 Apr 1997 04:26:33 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 9 Sender: daemon@net.bio.net Distribution: world Message-ID: <199704171127.UAA00577@mailgate.aist-nara.ac.jp> NNTP-Posting-Host: net.bio.net unsubscribe Yukio Kimata $BF`NI@hC<2J3X5;=QBg3X1!Bg3X!!(B $B0dEA;R650i8&5f%;%s%?! NNTP-Posting-Host: rhrz-ts1-p12.rhrz.uni-bonn.de X-Newsreader: Forte Free Agent 1.1/32.230 Dear colleagues, I am desperately looking for a quick and reasonable opportunity to sequence my purified proteins. These proteins are bound to a PVDF-membrane, so, that they can cut off easily for sequencing. What I want to know exactly is, who (maybe a company) is able to do the job for me and what will be the price for this. If there is someone out and know the answers please contact me by e-mail.My address is: unb13c@ibm.rhrz.uni-bonn.de. I appreciate every help. Thanks in advance, Mechthild From owner-fluorescent@net.bio.net Thu Apr 17 23:00:00 1997 Path: biosci!MPC186.MPIBPC.GWDG.DE!rbrock From: rbrock@MPC186.MPIBPC.GWDG.DE (Roland Brock) Newsgroups: bionet.molbio.proteins.fluorescent Subject: subscribe Date: 18 Apr 1997 07:21:26 -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 subscribe Roland Brock Max-Planck-Institute for Biophysical Chemistry Department of Molecular Biology Am Fassberg 37077 Goettingen Germany Ph.: +49-551-2011772 Fax.: +49-551-2011467 E-mail: rbrock@mpc186.mpibpc.gwdg.de From owner-fluorescent@net.bio.net Sat Apr 19 23:00:00 1997 Path: biosci!ihnp4.ucsd.edu!munnari.OZ.AU!news.ecn.uoknor.edu!feed1.news.erols.com!howland.erols.net!newsfeed.internetmci.com!news.map.com!usenet From: Barbara Foster Newsgroups: bionet.molbio.proteins.fluorescent Subject: New seminar: Optimizing Light Microscopy Date: Sun, 20 Apr 1997 21:37:14 -0700 Organization: Microscopy/Marketing & Education Lines: 59 Message-ID: <335AEEFA.6A56@mail.map.com> NNTP-Posting-Host: ppp47.map.com Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 8bit X-Mailer: Mozilla 2.01 (Win16; U) Seminar announcement: Optimizing Light Microscopy A one-day lecture/demonstration for anyone who uses or plans to use a microscope: students, teachers, medical technologists, clinicians, pathologists, and lab managers Six locations: Monday, April 28 - Marriott Marquis, NYC Tuesday, April 29 - Plainview Plaza-Hotel, Plainview, NY Thursday, May 1 - Tufts Medical School/Multi-Media Resource Center, Boston, MA Friday, May 2 - Tufts Medical School/Multi-Media Resource Center, Boston, MA Tuesday, May 6 - Holidome, Holyoke, MA (Greater Springfield area) * Wednesday, May 7 - Sheraton Hartford, Hartford, CT * * catered lunch available for an additional $15.50 Program: 1. A quick tour around the microscope 2. Alignment tips for reducing headaches, fatigue, and errors 3. Care, cleaning, and troubleshooting 4. Useful principles for understanding images 5. Quick, easy, and often free techniques for improving contrast (includes discussions of Phase and Hoffman Modulation Contrast) 6. Advanced techniques (Fluorescence and DIC) 7. Becoming a better consumer: matching your microscope to your applications 8. The Video connection: cameras, computers, and your microscope Fees: $115 if received before 4/18/97; $125 if received after that date. $50 discount on tuition for third person registered from the same facility. Free with your tuition: “Optimizing Light Microscopy for Biological and Clinical Laboratories” Over 220 pages of helpful tips, quick experiments, and new ideas for getting the best from your microscope (ASCLS/Kendall-Hunt, 1997) For further details, contact : Dr. Kenneth Piel or Barbara Foster Microscopy/Microscopy Education (MME) 53 Eton Street Springfield, MA 01108 Phone: (413)746-6931 Fax: (413)746-9311 email: mme@map.com To register: Fax or mail the form below to the MME office Name: _________________________________________________________________ School/Hospital/ Company:______________________________________________ Address: ________________________________________________________________ City/State/Zip: ___________________________________________________________ Phone: ____________________Fax: _______________email: __________________ Check enclosed (payable to MME) for: _________________ [ ] tuition only [ ] tuition plus lunch [ ] VISA [ ] Master Card Name on credit card: __________________________________________________ Card #: ___________________________________ Expiration date: ___________ From owner-fluorescent@net.bio.net Sat Apr 19 23:00:00 1997 Path: biosci!KUSUMIB.C.U-TOKYO.AC.JP!ike From: ike@KUSUMIB.C.U-TOKYO.AC.JP (Hiroshi Ike) Newsgroups: bionet.molbio.proteins.fluorescent Subject: unsubscribe Date: 20 Apr 1997 19:57:14 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 3 Sender: daemon@net.bio.net Distribution: world Message-ID: <9704210257.AA22845@biol1.bio.nagoya-u.ac.jp> NNTP-Posting-Host: net.bio.net unsubscribe From owner-fluorescent@net.bio.net Sat Apr 19 23:00:00 1997 Path: biosci!ihnp4.ucsd.edu!munnari.OZ.AU!news.ecn.uoknor.edu!feed1.news.erols.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!europa.clark.net!newsfeed2!usenet.logical.net!news.dal.ca!newsflash.concordia.ca!feed.umontreal.ca!usenet From: Guy Tremblay Newsgroups: bionet.molbio.proteins.fluorescent Subject: GFP expression in E. coli Date: Sun, 20 Apr 1997 11:10:05 -0400 Organization: Universite de Montreal Lines: 17 Distribution: world Message-ID: <335A31CD.53@medcn.umontreal.ca> Reply-To: tremblgu@medcn.umontreal.ca NNTP-Posting-Host: med07.medcn.umontreal.ca Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0Gold (Win95; I) Hi, I am interested in having an E. coli strain transduced with the GFP in the chromosome. Anybody has this? Also, I would like to know if anybody has problems transforming GFP into E. coli, on the pGFP plasmid. One student in our lab said he was only able to transform it into E.coli when he grew the bacteria on low salt agars. That surprises me and I'm curious if someone else encounters this problem. Any hints on GFP expression in E. coli is welcomed anyways. Thanks in advance and have a nice day, ----------------------------------------------------------------------- Guy Tremblay tremblgu@medcn.umontreal.ca From owner-fluorescent@net.bio.net Sun Apr 20 23:00:00 1997 Path: biosci!ACPUB.DUKE.EDU!ksub From: ksub@ACPUB.DUKE.EDU (Kala Subramanian) Newsgroups: bionet.molbio.proteins.fluorescent Subject: Re: unsubscribe Date: 21 Apr 1997 10:47:04 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 2 Sender: daemon@net.bio.net Distribution: world Message-ID: References: NNTP-Posting-Host: net.bio.net unsubscribe From owner-fluorescent@net.bio.net Sun Apr 20 23:00:00 1997 Path: biosci!bio.nagoya-u.ac.jp!ike From: ike@bio.nagoya-u.ac.jp (Hiroshi Ike) Newsgroups: bionet.molbio.proteins.fluorescent Subject: subscribe Date: 21 Apr 1997 02:55:17 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 2 Sender: daemon@net.bio.net Distribution: world Message-ID: <9704210932.AA25232@biol1.bio.nagoya-u.ac.jp> NNTP-Posting-Host: net.bio.net subscribe From owner-fluorescent@net.bio.net Sun Apr 20 23:00:00 1997 Path: biosci!SKI.MSKCC.ORG!f-parlati From: f-parlati@SKI.MSKCC.ORG (frank parlati) Newsgroups: bionet.molbio.proteins.fluorescent Subject: unsubscribe Date: 21 Apr 1997 10:07:31 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 3 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net unsubscribe From owner-fluorescent@net.bio.net Sun Apr 20 23:00:00 1997 Path: biosci!BIOCHEM.WISC.EDU!SBARROWS From: SBARROWS@BIOCHEM.WISC.EDU Newsgroups: bionet.molbio.proteins.fluorescent Subject: Forwarded: unsubscribe Date: 21 Apr 1997 09:34:33 -0700 Organization: Department of Biochemistry Lines: 10 Sender: daemon@net.bio.net Distribution: world Message-ID: <11B9C256E19@biochem.wisc.edu> NNTP-Posting-Host: net.bio.net To: fluorpro@net.bio.net From: sbarrows@biochem.wisc.edu Subject: unsubscribe Date: 20 Apr 1997 19:57:14 -0700 unsubscribe From owner-fluorescent@net.bio.net Sun Apr 20 23:00:00 1997 Path: biosci!aix.or.jp!aki-yama From: aki-yama@aix.or.jp ("yamamoto, aki") Newsgroups: bionet.molbio.proteins.fluorescent Subject: subscribe Date: 21 Apr 1997 08:07:56 -0700 Organization: ASCII Lines: 1 Sender: daemon@net.bio.net Distribution: world Message-ID: <335B8394.5E98@mailgw.aix.or.jp> Reply-To: aki-yama@aix.or.jp NNTP-Posting-Host: net.bio.net subscribe From owner-fluorescent@net.bio.net Sun Apr 20 23:00:00 1997 Path: biosci!TYRONE.WUSTL.EDU!suja From: suja@TYRONE.WUSTL.EDU (suja) Newsgroups: bionet.molbio.proteins.fluorescent Subject: unsubscribe Date: 21 Apr 1997 07:35:22 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 2 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net unsubscribe From owner-fluorescent@net.bio.net Sun Apr 20 23:00:00 1997 Path: biosci!U.WASHINGTON.EDU!moser From: moser@U.WASHINGTON.EDU ("'Mike' Michael J. Moser") Newsgroups: bionet.molbio.proteins.fluorescent Subject: Re: unsubscribe (for people who are to lazy to read the FAQ) Date: 21 Apr 1997 12:20:44 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 58 Sender: daemon@net.bio.net Distribution: world Message-ID: References: NNTP-Posting-Host: net.bio.net It seems that many of the postings lately have been of the above nature. Therefore I append a portion of the BioSci info file located at: ftp://net.bio.net/pub/BIOSCI/doc/biosci-us.infosheet Read this and you can unsubscribe yourself instead of bothering everybody else on the list! Info from file follows: ------------------------------------------------------------------------- !!! PLEASE BE SURE to send all subscribe or unsubscribe commands only to !!! the biosci-server@net.bio.net address and not to the newsgroup posting !!! addresses where they will bother all of the readers! Your e-mail address is obtained automatically from the header of your mail message and should NOT be included on the subscribe/unsubscribe command line unless you need to sign up at a different address than the one you are currently using. Such requests must be handled manually by us and create extra work. (Also you would be surprised at how many people list their e-mail addresses incorrectly which then results in nondelivery of mail, so please let our server obtain this information automatically from your mail header.) Multiple commands may be placed on separate lines in the same mail message, i.e., you can include all of your subscribe and/or unsubscribe requests in one mail message as long as each is on a separate line. If you put multiple commands in your mail message, please put an end command as the last in your list of commands. This helps avoid sending to the server any signatures that might be automatically included in your mail messages. Here is a sample subscription message. The address public@state-u.edu would be automatically added to the BIONEWS and GRASSES-SCIENCE mailing lists as the result of this message. Note that the listname for GRASSES-SCIENCE is "grasses" as derived from the address portion to the left of the @ sign in the mailing address for GRASSES-SCIENCE (see address table further below). ---------------------------------------------------------------------- To: biosci-server@net.bio.net From: J.Q. Public Subject: subscribe bionews subscribe grasses end ---------------------------------------------------------------------- Happy (un)subscribing Mike Moser Tel: 206-616-7391 UW Department of Pathology FAX: 206-543-3644 Box 357470 moser@u.washington.edu Seattle, WA 98195 http://weber.u.washington.edu/~moser From owner-fluorescent@net.bio.net Mon Apr 21 23:00:00 1997 Path: biosci!ihnp4.ucsd.edu!munnari.OZ.AU!news.ecn.uoknor.edu!feed1.news.erols.com!news.maxwell.syr.edu!news-xfer.cybernet.dk!news.kolumbus.fi!news.funet.fi!news.abo.fi!not-for-mail From: amikhail@news.abo.fi (Andrei Mikhailov BTC) Newsgroups: bionet.molbio.proteins.fluorescent Subject: subscribe Date: 22 Apr 1997 04:45:44 GMT Organization: Abo Akademi University Lines: 1 Message-ID: <5jhfpo$bsl@josie.abo.fi> NNTP-Posting-Host: aton.abo.fi X-Newsreader: TIN [UNIX 1.3 950520BETA PL0] do it immediately! From owner-fluorescent@net.bio.net Mon Apr 21 23:00:00 1997 Path: biosci!PANDA.UCHC.EDU!skwon From: skwon@PANDA.UCHC.EDU (Sunjong Kwon) Newsgroups: bionet.molbio.proteins.fluorescent Subject: BFP Date: 22 Apr 1997 08:20:25 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 20 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Does anyone know if there exists a commercial source of the blue-shifted mutant of GFP (Y66H) in a vector ready for expression in eukariotic cells? Blue fluorescent protein (BFP) vector for transfection of mammalian cells is available from Quantum Biotechnologies Inc. Sunjong Kwon Department of Biochemistry The University of Connecticut Health Center Farmington, CT. 06030 Tel:860-679-2106 Fax:860-679-3408 e-mail:skwon@panda.uchc.edu From owner-fluorescent@net.bio.net Mon Apr 21 23:00:00 1997 Path: biosci!SUN.BQ.UB.ES!ferrer From: ferrer@SUN.BQ.UB.ES (Joan Carles Ferrer) Newsgroups: bionet.molbio.proteins.fluorescent Subject: (none) Date: 22 Apr 1997 04:34:44 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 5 Sender: daemon@net.bio.net Distribution: world Message-ID: <199704222034.NAA05369@sun.bq.ub.es> NNTP-Posting-Host: net.bio.net Does anyone know if there exists a commercial source of the blue-shifted mutant of GFP (Y66H) in a vector ready for expression in eukariotic cells? From owner-fluorescent@net.bio.net Tue Apr 22 23:00:00 1997 Path: biosci!WAM.UMD.EDU!jrhens From: jrhens@WAM.UMD.EDU ("Julie R. Hens") Newsgroups: bionet.molbio.proteins.fluorescent Subject: Re: (none) Date: 23 Apr 1997 06:24:00 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 15 Sender: daemon@net.bio.net Distribution: world Message-ID: References: <199704222034.NAA05369@sun.bq.ub.es> NNTP-Posting-Host: net.bio.net Joan, my guess is you probably can get it from Clontech regards Julie On 22 Apr 1997, Joan Carles Ferrer wrote: > > Does anyone know if there exists a commercial source of > the blue-shifted mutant of GFP (Y66H) in a vector ready > for expression in eukariotic cells? > > > From owner-fluorescent@net.bio.net Tue Apr 22 23:00:00 1997 Path: biosci!aix.or.jp!aki-yama From: aki-yama@aix.or.jp ("yamamoto, aki") Newsgroups: bionet.molbio.proteins.fluorescent Subject: subscribe Date: 23 Apr 1997 08:44:59 -0700 Organization: ASCII Lines: 1 Sender: daemon@net.bio.net Distribution: world Message-ID: <335E2F4E.4DD5@mailgw.aix.or.jp> Reply-To: aki-yama@aix.or.jp NNTP-Posting-Host: net.bio.net subscribe bionet.molbio.proteins.fluorescent From owner-fluorescent@net.bio.net Wed Apr 23 23:00:00 1997 Path: biosci!ihnp4.ucsd.edu!munnari.OZ.AU!news.ecn.uoknor.edu!feed1.news.erols.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!newsfeed.nacamar.de!blackbush.xlink.net!news-ge.switch.ch!serra.unipi.it!newsserver.cilea.it!oracle.csi.unimi.it!usenet From: clsmteam@imiucca.csi.unimi.it (Paolo Castano) Newsgroups: bionet.molbio.proteins.fluorescent Subject: PROGRAM OF IMMUNOFLUORESCENCE COURSE Date: Thu, 24 Apr 1997 15:46:14 GMT Organization: Istitute of Human Anatomy Lines: 124 Message-ID: <335f8035.31870491@news.csi.unimi.it> NNTP-Posting-Host: isdn14.csi.unimi.it X-Newsreader: Forte Free Agent 1.1/32.230 Advanced International Immunofluorescence Course Gargnano '97 (Italy) The Advanced International Immunofluorescence Course is a post-doctorate theoretical/practical course, with propedeutical lectures and practical stages on traditional and confocal immunofluorescence microscopy and image and ion analysis. The course will take place in Gargnano (Lake of Garda) from 7 to 10 October 1997. Further information and registration details will be found at the following Web address http://imiucca.csi.unimi.it/endomi/ACIF.html Program: Morning Tuesday, October 7, 1997 9.00-10.00 Registration of partecipants 10.00-10.45 AN INTRODUCTION TO IMMUNOCYTOCHEMISTRY AND IMMUNOFLUORESCENCE (I.Barajon) 10.45-11.15 Coffee Break 11.15-12.00 LIGHT SOURCES AND FLUOROCHROMES (G. Bottiroli) 12.00-12.45 THE USE OF FLUORESCENCE MICROSCOPE (C.Rumio) 13.00-14.30 Work lunch Afternoon 14.00-14.45 FLUORESCENCE PHOTOMICROGRAPHY (P.Castano) 14.45-16.00 Practical stages: Photomicrography 16.00-16.30 Coffee Break 16.30-17.30 Practical stages: Photomicrography Morning Wednesday, October 8, 1997 9.00 - 9.45 IMMUNOCYTOCHEMISTRY AGAINST IMMUNOFLUORESCENCE (C.J.F.Van Noorden) 9.45-10.30 APPLICATION OF DIGITAL IMAGE ANALYSIS TO FLUORESCENCE MICROSCOPY (A.Remuzzi) 10.30-11.00 Coffee Break 11.00-11.45 PRINCIPLES OF CONFOCAL MICROSCOPY (G.J.Brakenhoff) 11.45-12.30 GFP AND Ca 2+ ANALYSIS (R. Rizzuto) 12.30-14.00 Work Lunch Afternoon 14.30-16.00 Practical stages: Photomicrography, Confocal Microscopy, Ca Analysis, Image Analysis. 16.00-16.30 Coffee Break 16.30-17.30 Practical stages: Photomicrography, Confocal Microscopy, Ca Analysis, Image Analysis. Evening 21.00-23.00 Illustration and discussion of photomicrographies. Morning Thursday, October 9, 1997 9.00 - 9.45 IMMUNOFLUORESCENCE FOR CELL CULTURES (C.Tacchetti) 9.45-10.30 IMMUNOFLUORESCENCE ON THICK SAMPLES (S.Modina) 10.30-11.00 Coffee Break 11.00-11.45 MULTIPHOTON LASER SCANNING FLUORESCENCE MICROSCOPY (A.Dixon) 11.45-12.30 MULTIPLE FLUORESCENCE (A. Entwistle) 12.30-14.00 Work Lunch Afternoon 14.00-17.00 Visit to the Vittoriale of G.D'Annunzio 17.00-19.30 Practical stages: Photomicrography, Confocal Microscopy, Ca Analysis, Image Analysis. Evening 20.30 All-together Dinner in a typical Restaurant Morning Friday, October 10, 1997 9.00 - 9.45 CONFOCAL MICROSCOPY AND FLUORESCENCE(A.Entwistle) 9.45 -11.00 Round Table: THE MULTI-DIMENSIONAL MICROSCOPY (G.J.Brakenhoff, A.Entwistle, P.Castano, A.Remuzzi, C.J.F.Van Noorden) 11.00-11.30 Coffee Break 11.30-13.30 Practical stages: Photomicrography, Confocal Microscopy, Ca Analysis, Image Analysis. 13.30-14.30 Work Lunch Afternoon 14.30-17.00 Practical stages: Photomicrography, Confocal Microscopy, Ca Analysis, Image Analysis. 17.30 End of works. Thank you Paolo Castano From owner-fluorescent@net.bio.net Wed Apr 23 23:00:00 1997 Path: biosci!PICR.CR.MAN.AC.UK!mlbsds From: mlbsds@PICR.CR.MAN.AC.UK ("Simon D. Scott") Newsgroups: bionet.molbio.proteins.fluorescent Subject: GFP and Flow Cytometry Date: 24 Apr 1997 07:02:43 -0700 Organization: Paterson Institute for Cancer Research Lines: 5 Sender: daemon@net.bio.net Distribution: world Message-ID: <335FD90E.2B91@picr.cr.man.ac.uk> NNTP-Posting-Host: net.bio.net Hello. We're investigating the use of wild-type and mutant GFPs in live and fixed cells (e.g. MCF7 breast tumour line) and wish to quantitate using FLOW CYTOMETRY. We are counterstaining fixed cells with PI. 70% ethanol is the fixative. Any advice? - Simon Scott/Brian Marples PICR From owner-fluorescent@net.bio.net Wed Apr 23 23:00:00 1997 Path: biosci!PICR.CR.MAN.AC.UK!mlbsds From: mlbsds@PICR.CR.MAN.AC.UK ("Simon D. Scott") Newsgroups: bionet.molbio.proteins.fluorescent Subject: test Date: 24 Apr 1997 07:02:25 -0700 Organization: Paterson Institute for Cancer Research Lines: 1 Sender: daemon@net.bio.net Distribution: world Message-ID: <335FD8E4.392E@picr.cr.man.ac.uk> NNTP-Posting-Host: net.bio.net test message From owner-fluorescent@net.bio.net Wed Apr 23 23:00:00 1997 Path: biosci!PICR.CR.MAN.AC.UK!bmcahc From: bmcahc@PICR.CR.MAN.AC.UK (A Cavinder) Newsgroups: bionet.molbio.proteins.fluorescent Subject: test message Date: 24 Apr 1997 06:55:18 -0700 Organization: Paterson Institute For Cancer Research Lines: 1 Sender: daemon@net.bio.net Distribution: world Message-ID: <335FD6BD.7CCE@picr.cr.man.ac.uk> Reply-To: bmcahc@picr.cr.man.ac.uk NNTP-Posting-Host: net.bio.net testing netscape mail From owner-fluorescent@net.bio.net Wed Apr 23 23:00:00 1997 Path: biosci!BIOCH.OX.AC.UK!recchia From: recchia@BIOCH.OX.AC.UK (Gavin Recchia) Newsgroups: bionet.molbio.proteins.fluorescent Subject: GFP-tet fusions Date: 24 Apr 1997 02:59:55 -0700 Organization: University of Oxford Lines: 9 Sender: daemon@net.bio.net Distribution: world Message-ID: <335F3DBF.7C15@bioch.ox.ac.uk> NNTP-Posting-Host: net.bio.net I heard some time ago on the grapevine that GFP-tet repressor fusions were commercially available, yet I have been unable to find anyone who can confirm this, and now don't know where the info came from in the first place! Does anyone know if such clones are available? Or does anyone out there use such a thing, and would it be possible for me to get hold of some? I would be extremely grateful for any information anyone could give me. Thanks in anticipation, Gavin Recchia From owner-fluorescent@net.bio.net Wed Apr 23 23:00:00 1997 Path: biosci!U.WASHINGTON.EDU!moser From: moser@U.WASHINGTON.EDU ("'Mike' Michael J. Moser") Newsgroups: bionet.molbio.proteins.fluorescent Subject: Re: GFP and Flow Cytometry Date: 24 Apr 1997 11:08:29 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 23 Sender: daemon@net.bio.net Distribution: world Message-ID: References: <335FD90E.2B91@picr.cr.man.ac.uk> NNTP-Posting-Host: net.bio.net Simon Alcohol fixation will rapidly destroy GFP fluorescence. I use 1/20th volume of formalin for 5 minutes at RT. Cells will remain fluorescent for several days stored in PBS at 4 C. WT GFP is essentially useless. Human codon bias optimized S65T (or other enhanced) GFP is highly recommended for FACS w/ human cells. Mike Moser Tel: 206-616-7391 UW Department of Pathology FAX: 206-543-3644 Box 357470 moser@u.washington.edu Seattle, WA 98195 http://weber.u.washington.edu/~moser On 24 Apr 1997, Simon D. Scott wrote: > Hello. We're investigating the use of wild-type and mutant GFPs in live > and fixed cells (e.g. MCF7 breast tumour line) and wish to quantitate > using FLOW CYTOMETRY. We are counterstaining fixed cells with PI. 70% > ethanol is the fixative. > Any advice? - Simon Scott/Brian Marples PICR > > From owner-fluorescent@net.bio.net Thu Apr 24 23:00:00 1997 Path: biosci!ihnp4.ucsd.edu!munnari.OZ.AU!news.ecn.uoknor.edu!feed1.news.erols.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!gatech!usenet.ins.cwru.edu!po.CWRU.Edu!mjb10 From: mjb10@po.CWRU.Edu (Michael J. Bumbulis) Newsgroups: bionet.molbio.proteins.fluorescent Subject: Re: GFP expression in E. coli Date: 26 Apr 1997 03:01:33 GMT Organization: Case Western Reserve University, Cleveland, OH (USA) Lines: 19 Message-ID: <5jrr6e$at1@madeline.INS.CWRU.Edu> References: <335A31CD.53@medcn.umontreal.ca> Reply-To: mjb10@po.CWRU.Edu (Michael J. Bumbulis) NNTP-Posting-Host: christopher.ins.cwru.edu In a previous article, tremblgu@medcn.umontreal.ca (Guy Tremblay) says: >Hi, > >I am interested in having an E. coli strain transduced with the GFP in >the chromosome. Anybody has this? > >Also, I would like to know if anybody has problems transforming GFP into >E. coli, on the pGFP plasmid. One student in our lab said he was only >able to transform it into E.coli when he grew the bacteria on low salt >agars. That surprises me and I'm curious if someone else encounters this >problem. Using standard LB media, I had no problem at all in transforming E.coli with this plasmid. -- From owner-fluorescent@net.bio.net Thu Apr 24 23:00:00 1997 Path: biosci!ihnp4.ucsd.edu!munnari.OZ.AU!news.ecn.uoknor.edu!feed1.news.erols.com!news.maxwell.syr.edu!newsspool.doit.wisc.edu!news.doit.wisc.edu!news From: Joe Binder Newsgroups: bionet.molbio.proteins.fluorescent Subject: GFP in HeLa cells Date: Fri, 25 Apr 1997 14:23:38 -0600 Organization: University of Wisconsin, Madison Lines: 4 Message-ID: <336112CA.7A84@students.wisc.edu> NNTP-Posting-Host: thunderweasel.ahabs.wisc.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.0 (Macintosh; I; 68K) I have put GFP (ser65thr and ile167thr) into Mengo virus and am using fluorescent microscopy to visualize infected cells. I have seen low expression levels. Any advice on preparing/visualizing slides to see the protein better? From owner-fluorescent@net.bio.net Thu Apr 24 23:00:00 1997 Path: biosci!daresbury!uninett.no!news.uit.no!vi05.imb.fm.uit.no!user From: olems@fagmed.uit.no (ole morten seternes) Newsgroups: bionet.molbio.proteins.fluorescent Subject: Re: GFP-tet fusions Date: 25 Apr 1997 12:47:40 GMT Organization: university of tromsoe, IMB, Dep.of Virology Lines: 17 Distribution: world Message-ID: References: <335F3DBF.7C15@bioch.ox.ac.uk> NNTP-Posting-Host: vi05.imb.fm.uit.no In article <335F3DBF.7C15@bioch.ox.ac.uk>, recchia@BIOCH.OX.AC.UK (Gavin Recchia) wrote: > I heard some time ago on the grapevine that GFP-tet repressor fusions > were commercially available, yet I have been unable to find anyone who > can confirm this, and now don't know where the info came from in the > first place! Does anyone know if such clones are available? Or does > anyone out there use such a thing, and would it be possible for me to > get hold of some? I would be extremely grateful for any information > anyone could give me. > Thanks in anticipation, > Gavin Recchia Dear Gavin Check out clontech´s homepage: http://www.clontech.com Ole Morten Seternes From owner-fluorescent@net.bio.net Sun Apr 27 23:00:00 1997 Path: biosci!FMI.CH!ludin From: ludin@FMI.CH (Beat Ludin) Newsgroups: bionet.molbio.proteins.fluorescent Subject: Re: GFP in HeLa cells Date: 28 Apr 1997 00:03:52 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 24 Sender: daemon@net.bio.net Distribution: world Message-ID: <199704280700.JAA15216@fmi.ch> NNTP-Posting-Host: net.bio.net Joe Binder wrote: >I have put GFP (ser65thr and ile167thr) into Mengo virus and am using >fluorescent microscopy to visualize infected cells. I have seen low >expression levels. Any advice on preparing/visualizing slides to see >the protein better? 1. Using a low-riboflavin medium or a blanaced salt solution should lower the background. 2. Don't fix the cells to get the highest fluorescence output. 3. If fixation is needed, paraformaldehye seems to be the least damaging. Readjust the pH to 6.5 or higher. BTW, I'm not aware of a S65T/I167T mutant, what is its spectrum? Can you give me a reference? Beat ----------------------------------------------------- Dr. Beat Ludin, FMI, PO Box 2543, 4002 Basel, Switzerland Tel. +41 61 697 6697 / FAX +41 61 697 3976 Internet:ludin@fmi.ch / Compuserve:100102,1527 From owner-fluorescent@net.bio.net Sun Apr 27 23:00:00 1997 Path: biosci!CMB.BIOSCI.WAYNE.EDU!hivlab From: hivlab@CMB.BIOSCI.WAYNE.EDU ("A. S. Goustin Lab") Newsgroups: bionet.molbio.proteins.fluorescent Subject: gfp in Gene Therapy/a Stockton Press Journal Date: 28 Apr 1997 08:56:30 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 15 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net From the table of contents: Gene Therapy May 1997, Volume 4, Number 5 Table of Contents ---------------------------------------------------------------------- 493 Adenovirus-mediated expression of green fluorescent protein R de Martin, M Raidl, E Hofer and BR Binder ---------------------------------------------------------------------- From owner-fluorescent@net.bio.net Sun Apr 27 23:00:00 1997 Path: biosci!FMI.CH!ludin From: ludin@FMI.CH (Beat Ludin) Newsgroups: bionet.molbio.proteins.fluorescent Subject: Re: GFP in HeLa cells Date: 28 Apr 1997 07:33:33 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 22 Sender: daemon@net.bio.net Distribution: world Message-ID: <199704281430.QAA27001@fmi.ch> NNTP-Posting-Host: net.bio.net >>BTW, I'm not aware of a S65T/I167T mutant, what is its spectrum? Can you >>give me a reference? >Hi Beat, > > >The single mutants shift the excitation spectrum to a single peak near >475nm, with emission at 508nm IIRC... > Ok, seems like I got it wrong. I thought, Joe Binder was talking about a S65T/I167T double mutant. (I'm familiar with the single mutants). Beat ----------------------------------------------------- Dr. Beat Ludin, FMI, PO Box 2543, 4002 Basel, Switzerland Tel. +41 61 697 6697 / FAX +41 61 697 3976 Internet:ludin@fmi.ch / Compuserve:100102,1527 From owner-fluorescent@net.bio.net Sun Apr 27 23:00:00 1997 Path: biosci!fcs280s.ncifcrf.gov!cpk-news-feed1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!rill.news.pipex.net!pipex!server1.netnews.ja.net!warwick!lyra.csx.cam.ac.uk!drm21 From: drm21@mole.bio.cam.ac.uk (David Micklem) Newsgroups: bionet.molbio.proteins.fluorescent Subject: Re: GFP in HeLa cells Date: Mon, 28 Apr 1997 13:50:56 +0000 Organization: Wellcome/CRC Institute Lines: 41 Distribution: world Message-ID: References: <199704280700.JAA15216@fmi.ch> NNTP-Posting-Host: drm-mac1.welc.cam.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=ISO-8859-1 Content-Transfer-Encoding: 8bit X-Newsreader: Yet Another NewsWatcher 2.3.0 In article <199704280700.JAA15216@fmi.ch>, ludin@FMI.CH (Beat Ludin) wrote: >Joe Binder wrote: > >>I have put GFP (ser65thr and ile167thr) into Mengo virus and am using >>fluorescent microscopy to visualize infected cells. I have seen low >>expression levels. Any advice on preparing/visualizing slides to see >>the protein better? > >1. Using a low-riboflavin medium or a blanaced salt solution should lower >the background. >2. Don't fix the cells to get the highest fluorescence output. >3. If fixation is needed, paraformaldehye seems to be the least damaging. >Readjust the pH to 6.5 or higher. > >BTW, I'm not aware of a S65T/I167T mutant, what is its spectrum? Can you >give me a reference? > >Beat > Hi Beat, You might look at: 1. Heim R, Prasher DC, Tsien RY. Wavelength mutations and posttranslational auto oxidation of green fluorescent protein. Proc. Natl. Acad. Sci USA 1994, 91:12501-12504. 2. Heim R, Cubitt AB, Tsien RY. Improved green fluorescence. Nature 1995, 373:663-664. 3. Brand AH. GFP in Drosophila. Trends in Genetics 1995, 11:324-325. The single mutants shift the excitation spectrum to a single peak near 475nm, with emission at 508nm IIRC... David -- D.R.Micklem, Time flies like an arrow... Wellcome/CRC Institute, Fruit flies like a banana. Cambridge CB2 1QR, UK Email:drm21@mole.bio.cam.ac.uk Junk mail very very unwelcome. From owner-fluorescent@net.bio.net Mon Apr 28 23:00:00 1997 Path: biosci!daresbury!not-for-mail From: Newsgroups: bionet.molbio.proteins.fluorescent Subject: EGFP in S. cerevisiae Date: 29 Apr 1997 16:31:52 +0100 Lines: 22 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <5k5498$s1u@mserv1.dl.ac.uk> Original-To: fluorpro@dl.ac.uk Registered-Mail-Reply-Requested-By: bozic@britbio.co.uk EGFP in S. cerevisiae ---------------------- Hello, We are trying to express and detect EGFP protein in S. cerevisiae. Gene sequence has been checked, and Nothern Blot analysis indicates that the gene is transcribed, nevertheless, we are not able to detect any fluorescence (fluorescence microscope, FACS, Fluorometer). We decided to construct a strong positif control (EGFP cloned in a multi-copy plasmid, under control of a strong inducible promoter, and transformed in a protease deficient yeast strain), but as previously, no fluorescence is detected. Cells have been grown on liquid and solid medium, at 30C and at room temperature. Has anybody same problems to detect EGFP or has someone some indications which could help us ? Thank you for your help. C. BOZIC bozic@britbio.co.uk From owner-fluorescent@net.bio.net Mon Apr 28 23:00:00 1997 Path: biosci!U.WASHINGTON.EDU!moser From: moser@U.WASHINGTON.EDU ("'Mike' Michael J. Moser") Newsgroups: bionet.molbio.proteins.fluorescent Subject: Re: EGFP in S. cerevisiae Date: 29 Apr 1997 11:05:49 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 50 Sender: daemon@net.bio.net Distribution: world Message-ID: References: <5k5498$s1u@mserv1.dl.ac.uk> NNTP-Posting-Host: net.bio.net Dear C. Brovic, The EGFP sequence has been optimized for human expression by changing the codon usage to that prefered by mammalian cells. My guess is that your constructs aren't working because of this. My experience with the wt (A. victoria) cDNA and the S65T mutant (A. victoria codons) was that even single copy integrants of an S65T GFP-calmodulin fusion were easily detectable in both S. cerevisiae and S. pombe by fluorescence microscopy and by FACS. Cerevisiae cells containing a GAL driven S65T GFP construct were screaming. Pombe cells containing nmt driven S65T GFP were so bright they look like little "light bulbs". Brendan Cormack has found that the A. victoria cDNA is not expressed in C. albicans and he has constructed a yeast codon usage version of his FACS selected mutants that does express. Supposedly these versions give a further few-fold increase in budding yeast, but I haven't tried them myself. So EGFP is probably not thecDNA of choice for yeast. MIKE Mike Moser Tel: 206-616-7391 UW Department of Pathology FAX: 206-543-3644 Box 357470 moser@u.washington.edu Seattle, WA 98195 http://weber.u.washington.edu/~moser On 29 Apr 1997 bozic@britbio.co.uk wrote: > EGFP in S. cerevisiae > ---------------------- > > Hello, > > We are trying to express and detect EGFP protein in S. cerevisiae. > Gene sequence has been checked, and Nothern Blot analysis indicates > that the gene is transcribed, nevertheless, we are not able to > detect any fluorescence (fluorescence microscope, FACS, > Fluorometer). We decided to construct a strong positif control (EGFP > cloned in a multi-copy plasmid, under control of a strong inducible > promoter, and transformed in a protease deficient yeast strain), but > as previously, no fluorescence is detected. Cells have been grown on > liquid and solid medium, at 30C and at room temperature. > > Has anybody same problems to detect EGFP or has someone some > indications which could help us ? > > Thank you for your help. > > C. BOZIC > bozic@britbio.co.uk > > From owner-fluorescent@net.bio.net Mon Apr 28 23:00:00 1997 Path: biosci!CROP.UOGUELPH.CA!ACARLSON From: ACARLSON@CROP.UOGUELPH.CA ("Alvar Carlson") Newsgroups: bionet.molbio.proteins.fluorescent Subject: Replies to accumulation of GFP in plant nucleus Date: 29 Apr 1997 11:58:55 -0700 Organization: Crop Science, The Univ. of Guelph Lines: 39 Sender: daemon@net.bio.net Distribution: world Message-ID: <841530807C0@csnet.nw.uoguelph.ca> NNTP-Posting-Host: net.bio.net On April 14th I posted the following questions: > > I am attempting to use sGFP to visually select transgenic barley. >I am concerned that the accumulation of the GFP in the nucleus will >be detrimental to the development of transgenic plants. Has anyone >observed reduced transformation efficiency in plants when using GFP >or reduced fertility in any of the transgenic plants generated? >Furthermore, has anyone found that modified GFP, to exclude it from >the nucleus, increases the transformation efficiency? ......................................... From: David Galbraith We have expressed GFP transgenically targeted to the nucleus in tobacco and have seen no evidence of toxicity. Plants have not gone to the next generation yet. From: sjdavis1@students.wisc.edu (Seth J. Davis) WT GFP is toxic, and interferes with transformation efficiency. Modified GFPs (smGFP or mGFP5) appear to be less toxic in regenerating plants. mGFP5, which is ER localized (not in the nucleus) might reduce the toxic effects of GFP. This last point is not clear; is making GFP soluble or is localizing GFP to the ER increasing transformation efficiency? This has not been tested. From: krs1@mrc-lmb.cam.ac.uk (Kirby Siemering) We found it necessary to remove GFP from the nucleus by localising it to the ER in order to successfully regenerate bright and healthy Arabidopsis plants. See Haseloff et al PNAS 1997 (94) 2122-2127 and http://brindabella.mrc-lmb.cam.ac.uk/ for details. ........................... Thanks for the replies, Alvar Carlson University of Guelph From owner-fluorescent@net.bio.net Tue Apr 29 23:00:00 1997 Path: biosci!ihnp4.ucsd.edu!munnari.OZ.AU!news.ecn.uoknor.edu!feed1.news.erols.com!insync!enews.sgi.com!news.corp.sgi.com!news.sgi.com!sdd.hp.com!night.primate.wisc.edu!newsspool.doit.wisc.edu!news.doit.wisc.edu!news From: Joe Binder Newsgroups: bionet.molbio.proteins.fluorescent Subject: Re: GFP in HeLa cells Date: Wed, 30 Apr 1997 15:52:08 -0600 Organization: University of Wisconsin, Madison Lines: 18 Message-ID: <3367BF08.C48@students.wisc.edu> References: <199704281430.QAA27001@fmi.ch> NNTP-Posting-Host: thunderweasel.ahabs.wisc.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.0 (Macintosh; I; 68K) To: Beat Ludin You were right. I do have the double mutant. Unfortunately, I don't have much information about it and attempts to find out more haven't been answered. Here's what I do know: In C.elegans I believe, the double mutant appeared to be brighter than the original GFP or the ind. mutants alone, when driven under the unc-25 and vab-3 promoters. They have broader emission and excitation spectra (rhodoamine filters still show flourescence, although not as bright as fluor.). From what I know, this variant works well in elegans. I have had some trouble in HeLa, but I believe it may be the temp (37 for HeLa, 15-25 for elegans) and the quality of the scope. I plan to alter the conditions a little and fluorescence hopefully will improve. Yishi Jin from UC-SC created the construct. However, it has not been well characterized and no other info about it has been provided. From owner-fluorescent@net.bio.net Tue Apr 29 23:00:00 1997 Path: biosci!fcs280s.ncifcrf.gov!cpk-news-feed1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!newsfeed.nacamar.de!uunet!in1.uu.net!128.6.21.17!dziuxsolim.rutgers.edu!amenti.rutgers.edu!not-for-mail From: meton@rci.rutgers.edu (Daniel Gonzalez) Newsgroups: bionet.molbio.proteins.fluorescent Subject: International GFP Symposium Date: 30 Apr 1997 19:49:27 -0400 Organization: Rutgers University Lines: 10 Message-ID: <5k8lq7$crg@amenti.rutgers.edu> NNTP-Posting-Host: amenti.rutgers.edu Summary: Update on the International GFP Symposium Keywords: Green Fluorescent Protein GFP The International Green-Fluorescent Protein Symposium Call for speakers The Center for Research and Education in Bioluminescence (CREBB), a component of Rutgers University Cook College Campus, in conjunction with the Cook College Office of Continuing Professional Education, is holding an international symposium on Green-Fluorescent Protein, October 18-22, 1997 in New Brunswick, New Jersey, USA. Principals interested in presenting an oral presentation should contact CREBB by e-mail at meton@rci.rutgers.edu (Daniel Gonzalez). I will send those interested a calender of events that will be presented at the symposium. All facets of GFP biochemistry and applications will be discussed. Please include your regular (snail) mail for more detailed information as it becomes available. We have already 18 notables involved in GFP research who have volunteered to speak. Important and groundbreaking work in the field will be presented as well as a retrospective of the history of GFP and its use as a marker for gene expression. We are still seeking attendees, and suggestions for topics, as we are still in the planning stages. Additional announcements will follow and soon we will have a web site up. A registration packet will be produced and sent to those on our mailing lists. The symposia will be coordinated by Dr. William W. Ward, director of CREBB and Rutgers University Professor, who has been involved in GFP research for the last twenty years and will be attended by the leading researchers in the field. From owner-fluorescent@net.bio.net Tue Apr 29 23:00:00 1997 Path: biosci!FMI.CH!ludin From: ludin@FMI.CH (Beat Ludin) Newsgroups: bionet.molbio.proteins.fluorescent Subject: Re: eGFP deletion Date: 30 Apr 1997 07:57:39 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 23 Sender: daemon@net.bio.net Distribution: world Message-ID: <199704301454.QAA09937@fmi.ch> NNTP-Posting-Host: net.bio.net SAINTIGNY Yannick 161011 wrote: > Does any one known what will be the minimal deletion in the 3' end of >the eGFP coding sequence to knock-out the transcription and/or the >traduction and/or the fluorescence of the eGFP, in mammalian cell >transfected with the peGFP-C3 vector ( without deleting the three STOPs >codon). What happen if we remove the three last amino-acid of the eGFP ? As far as I remember (Roger Tsien mentioned it at the last ASCB meeting), one can delete a maximum of about 5 AA on the c-terminus of GFP and still get a fluorescent protein. The situation should be the same for eGFP. Beat ----------------------------------------------------- Dr. Beat Ludin, FMI, PO Box 2543, 4002 Basel, Switzerland Tel. +41 61 697 6697 / FAX +41 61 697 3976 Internet:ludin@fmi.ch / Compuserve:100102,1527 From owner-fluorescent@net.bio.net Tue Apr 29 23:00:00 1997 Path: biosci!U.WASHINGTON.EDU!toddman From: toddman@U.WASHINGTON.EDU ("R. Maney") Newsgroups: bionet.molbio.proteins.fluorescent Subject: Re: GFP fusion protein in CHO Date: 30 Apr 1997 10:11:22 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 35 Sender: daemon@net.bio.net Distribution: world Message-ID: References: NNTP-Posting-Host: net.bio.net with our experience, GFP alone in CHO cells does not aggregate in any specific structure but localizes to both the cytoplasm and nucleus uniformly. One note: our pictures seem to show slightly darker staining in the nucleus than the cytoplasm but this could be due to cell geometry rather than a property of GFP. GFP, given its small size, is expected to enter the nucleus via free diffusion. todd On 30 Apr 1997, Beatrice Marqueze wrote: > I have constructed a fusion protein with GPF using Clontech vector > pEGFP-N1. My goal is to study in CHO cells the distribution of the fusion > protein in the different sub-cellular compartments. > > Does anybody know where GFP alone is distibuted when transfected in CHO, in > the cytoplasm, in the nucleus ? Could the fusion protein be wrongly > localized in the GFP compartments? > > > .............................................................................. > Beatrice Marqueze-Pouey > INSERM U 374/464, Institut Federatif Jean Roche, Faculte de Medecine-Nord, > 13916 Marseille Cedex 20, France. > Tel (33) 4 91 69 88 32, Fax (33) 4 91 09 05 06, > E-mail: marqueze@jean-roche.univ-mrs.fr > ............................................................................ > .. > > > > From owner-fluorescent@net.bio.net Tue Apr 29 23:00:00 1997 Path: biosci!daresbury!lyra.csx.cam.ac.uk!nadgee.mrc-lmb.cam.ac.uk!user From: krs1@mrc-lmb.cam.ac.uk (Kirby Siemering) Newsgroups: bionet.molbio.proteins.fluorescent Subject: Re: Replies to accumulation of GFP in plant nucleus Date: Wed, 30 Apr 1997 12:18:16 +0100 Organization: MRC Laboratory of Molecular Biology Lines: 68 Distribution: world Message-ID: References: <841530807C0@csnet.nw.uoguelph.ca> NNTP-Posting-Host: nadgee.mrc-lmb.cam.ac.uk In article <841530807C0@csnet.nw.uoguelph.ca>, ACARLSON@CROP.UOGUELPH.CA ("Alvar Carlson") wrote: > On April 14th I posted the following questions: > > > > I am attempting to use sGFP to visually select transgenic barley. > >I am concerned that the accumulation of the GFP in the nucleus will > >be detrimental to the development of transgenic plants. Has anyone > >observed reduced transformation efficiency in plants when using GFP > >or reduced fertility in any of the transgenic plants generated? > >Furthermore, has anyone found that modified GFP, to exclude it from > >the nucleus, increases the transformation efficiency? > ......................................... > > > From: David Galbraith > > We have expressed GFP transgenically targeted to the nucleus in tobacco and > have seen no evidence of toxicity. Plants have not gone to the next > generation yet. > > From: sjdavis1@students.wisc.edu (Seth J. Davis) > > WT GFP is toxic, and interferes with transformation efficiency. Modified > GFPs (smGFP or mGFP5) appear to be less toxic in regenerating plants. > mGFP5, which is ER localized (not in the nucleus) might reduce the toxic > effects of GFP. This last point is not clear; is making GFP soluble or is > localizing GFP to the ER increasing transformation efficiency? This has > not been tested. > > From: krs1@mrc-lmb.cam.ac.uk (Kirby Siemering) > > We found it necessary to remove GFP from the nucleus by localising it to > the ER in order to successfully regenerate bright and healthy Arabidopsis > plants. See Haseloff et al PNAS 1997 (94) 2122-2127 and > http://brindabella.mrc-lmb.cam.ac.uk/ > for details. A couple of comments. Firstly, wt GFP is largely insoluble when expressed in bacteria at 37 degrees. However, it is >90% soluble when expressed at 25 degrees. The insolubility at higher temperatures is due to a temperature-dependent lesion in the folding of the apoprotein (Siemering et al. 1996 Curr Biol 6 1653-1663). These results suggest that wt GFP should already be soluble when expressed in plant cells at around 23 degrees, although this has not been proven one way or the other. Secondly, we did not find wt GFP to result in a lower transformation efficiency per se. In fact, we were able to consistently produce very bright callus using wt GFP. Our problem was that we were consistently unable to regenerate this brightest callus into bright and fertile plants. Localisation of GFP to the ER has appeared to cure this problem with very bright callus being produced which can successfully be regenerated into fertile plants that show high levels of fluorescence. See the above-mentioned PNAS reference for details and the web site for an image of a mgfp5-ER expressing adult plant under UV lamp illumination. Cheers, Kirby Dr. Kirby Siemering Division of Cell Biology MRC Laboratory of Molecular Biology Addenbrookes Hospital, Hills Road. Cambridge. CB2 2QH England. Ph. (01223) 402024 FAX (01223) 402008 email krs1@mrc-lmb.cam.ac.uk From owner-fluorescent@net.bio.net Tue Apr 29 23:00:00 1997 Path: biosci!daresbury!not-for-mail From: SAINTIGNY Yannick 161011 Newsgroups: bionet.molbio.proteins.fluorescent Subject: eGFP deletion Date: 30 Apr 1997 11:55:22 +0100 Lines: 12 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <5k78eq$qrl@mserv1.dl.ac.uk> MIME-Version: 1.0 Original-To: "'Liste EGFP'" HeIlo, I'am new in the eGFP field, and I will ask the following question : Does any one known what will be the minimal deletion in the 3' end of the eGFP coding sequence to knock-out the transcription and/or the traduction and/or the fluorescence of the eGFP, in mammalian cell transfected with the peGFP-C3 vector ( without deleting the three STOPs codon). What happen if we remove the three last amino-acid of the eGFP ? Thank, I' m waiting for replies. Franck AMIOT From owner-fluorescent@net.bio.net Tue Apr 29 23:00:00 1997 Path: biosci!jean-roche.univ-mrs.fr!marqueze From: marqueze@jean-roche.univ-mrs.fr (Beatrice Marqueze) Newsgroups: bionet.molbio.proteins.fluorescent Subject: GFP fusion protein in CHO Date: 30 Apr 1997 03:06:10 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 19 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net I have constructed a fusion protein with GPF using Clontech vector pEGFP-N1. My goal is to study in CHO cells the distribution of the fusion protein in the different sub-cellular compartments. Does anybody know where GFP alone is distibuted when transfected in CHO, in the cytoplasm, in the nucleus ? Could the fusion protein be wrongly localized in the GFP compartments? .............................................................................. Beatrice Marqueze-Pouey INSERM U 374/464, Institut Federatif Jean Roche, Faculte de Medecine-Nord, 13916 Marseille Cedex 20, France. Tel (33) 4 91 69 88 32, Fax (33) 4 91 09 05 06, E-mail: marqueze@jean-roche.univ-mrs.fr ............................................................................ .. From owner-fluorescent@net.bio.net Tue Apr 29 23:00:00 1997 Path: biosci!bcm.tmc.edu!cs.utexas.edu!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!uunet!in2.uu.net!208.198.64.1!netnews.sbphrd.com!news From: Simon_Q_Rice Newsgroups: bionet.molbio.proteins.fluorescent Subject: Extra Not-1 site in pEGFP Date: Wed, 30 Apr 1997 10:55:55 -0700 Organization: SmithKline Beecham Lines: 18 Message-ID: <336787AB.336D@sbphrd.com> NNTP-Posting-Host: hhd897.ha.uk.sbphrd.com Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01 (Win16; I) -Hi, I attempted to use the "unique" Not-l site in the MCS of Clontech's pEGFP. However, it would appear there are two such sites within this plasmid. Has anyone else found this problem? Does anyone have pEGFP with a unique Not-1 please? The purpose of this e-mail is informative. Clontech have been made aware and to their credit their UK distributors have refunded the cost of purchase.- Cheers Simon The opinions expressed in this communication are my own, and do not necessarily reflect those of my employer. From owner-fluorescent@net.bio.net Tue Apr 29 23:00:00 1997 Path: biosci!FMI.CH!ludin From: ludin@FMI.CH (Beat Ludin) Newsgroups: bionet.molbio.proteins.fluorescent Subject: Re: GFP fusion protein in CHO Date: 30 Apr 1997 07:57:37 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 34 Sender: daemon@net.bio.net Distribution: world Message-ID: <199704301454.QAA09880@fmi.ch> NNTP-Posting-Host: net.bio.net Beatrice Marqueze wrote: >I have constructed a fusion protein with GPF using Clontech vector >pEGFP-N1. My goal is to study in CHO cells the distribution of the fusion >protein in the different sub-cellular compartments. > >Does anybody know where GFP alone is distibuted when transfected in CHO, in >the cytoplasm, in the nucleus ? Yes, i.e. both in the cytoplasm and in the nucleus with a slight tendency to accumulate into the nucleus. > Could the fusion protein be wrongly >localized in the GFP compartments? We have made a number of GFP fusion proteins so far, and in none of the cases we have observed a mislocalisation. I haven't heard of something like that happening either. But it's certainly conceivable. For example, a protein which normally enters the nucleus could become to big to do so because of the increased size of the fusion protein. Or a cytoskeletal protein may show even cytoplasmic distribution because the fusion with GFP has abolished its binding ability. But all the current evidence I know of points against an active GFP-mediated relocalisation. So my advice is: go ahead. Beat ----------------------------------------------------- Dr. Beat Ludin, FMI, PO Box 2543, 4002 Basel, Switzerland Tel. +41 61 697 6697 / FAX +41 61 697 3976 Internet:ludin@fmi.ch / Compuserve:100102,1527 From owner-fluorescent@net.bio.net Tue Apr 29 23:00:00 1997 Path: biosci!UCSD.EDU!rtsien From: rtsien@UCSD.EDU ("Roger Y. Tsien") Newsgroups: bionet.molbio.proteins.fluorescent Subject: Re: eGFP deletion Date: 30 Apr 1997 21:17:30 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 44 Sender: daemon@net.bio.net Distribution: world Message-ID: <3.0.1.32.19970430204823.006d473c@sdcc12.ucsd.edu> References: <199704301454.QAA09937@fmi.ch> NNTP-Posting-Host: net.bio.net At 07:57 AM 4/30/97 -0700, you wrote: >SAINTIGNY Yannick 161011 wrote: > >> Does any one known what will be the minimal deletion in the 3' end of >>the eGFP coding sequence to knock-out the transcription and/or the >>traduction and/or the fluorescence of the eGFP, in mammalian cell >>transfected with the peGFP-C3 vector ( without deleting the three STOPs >>codon). What happen if we remove the three last amino-acid of the eGFP ? > >As far as I remember (Roger Tsien mentioned it at the last ASCB meeting), >one can delete a maximum of about 5 AA on the c-terminus of GFP and still >get a fluorescent protein. The situation should be the same for eGFP. > >Beat > I don't recall giving a figure of 5 AA at the C-terminus of GFP in my talk, but if I did, I would like to correct any such off-the-cuff remark and cite the references in which the real data is published, which anyone interested should read for themselves: Dopf and Horiagan (1996) produced a family of genetic truncations from the N- and C-termini. The N-terminal methionine could be replaced by a polyhistidine tag, but deletion of residues 2-8 prevented fluorescence or chromophore development. The C-terminus was slightly more tolerant, in that 6 but not 13 residues could be eliminated. These narrow limits are in good agreement with the crystal structure (Ormo et al. 1996), in which Met1 and residues 230-238 are too disordered to be located. Dopf, J. and Horiagon, T. (1996) Deletion mapping of Aequorea victoria green fluorescent protein. Gene 173, 39-44. Ormo, M., Cubitt, A.B., Kallio, K., Gross, L.A., Tsien, R.Y. and Remington, S.J. (1996) Crystal structure of the Aequorea victoria green fluorescent protein. Science 273, 1392-1395. Roger Y. Tsien rtsien@ucsd.edu tel. +1(619)534-4891, fax +1(619)534-5270 Howard Hughes Med. Inst., Univ. Calif. San Diego, La Jolla, CA 92093-0647 From owner-fluorescent@net.bio.net Wed Apr 30 23:00:00 1997 Path: biosci!daresbury!not-for-mail From: Peter Corish Newsgroups: bionet.molbio.proteins.fluorescent Subject: Fluorescent beads for FACS Date: 1 May 1997 11:41:47 +0100 Lines: 24 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <5k9s1b$3d4@mserv1.dl.ac.uk> X-Sender: pcor@topaz MIME-Version: 1.0 Original-To: Fluorescent newsgroup Dear all, Are there any commercial suppliers of GFP-coated beads suitable for quantification and calibration in FACS analysis? We have some FITC beads but need GFP of any sort. Alternatively, does anyone know how to crosslink purified GFP (such as the one Clontech sell) onto beads yourself and is it possible to quantify these accurately? Any help gratefully received, Many thanks, Pete ______________________________________________________________________________ Peter Corish CRC Chromosome Molecular Biology Group Department of Biochemistry University of Oxford Oxford,U.K. OX1 3QU Tel: +44 (0)1865 275222 Fax: +44 (0)1865 275283 _____________________________________________________________________________ From owner-fluorescent@net.bio.net Wed Apr 30 23:00:00 1997 Path: biosci!COMPUSERVE.COM!100102.1527 From: 100102.1527@COMPUSERVE.COM (Beat Ludin) Newsgroups: bionet.molbio.proteins.fluorescent Subject: Re: eGFP deletion Date: 1 May 1997 14:09:48 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 28 Sender: daemon@net.bio.net Distribution: world Message-ID: <970501210937_100102.1527_EHK55-1@CompuServe.COM> NNTP-Posting-Host: net.bio.net Beat Ludin wrote: >>As far as I remember (Roger Tsien mentioned it at the last ASCB meeting), >one can delete a maximum of about 5 AA on the c-terminus of GFP... "Roger Y. Tsien" wrote: >I don't recall giving a figure of 5 AA at the C-terminus of GFP in my talk,... My sincere apologies in case that I have cited you incorrectly - I recognize that I should not cite other people from the top of my head w/o being absolutely sure about the accuracy of the information - and thanks a lot for clarifying the issue. Sincerely, Beat Ludin ---------------------------------------------- Beat Ludin, Ph.D., Friedrich Miescher Institut Maulbeerstr. 66, 4058 Basel, Switzerland Fon +41 61 697 6697, Fax +41 61 697 3976