From jjsun from MIT.EDU Wed Jul 8 22:05:29 2009 From: jjsun from MIT.EDU (Jingjing Sun) Date: Thu Jul 9 04:26:08 2009 Subject: [Yeast] homologous recombination in yeast Message-ID: <4A555E79.80108@mit.edu> Dear Yeast Netters, I have some questions about homologous recombination in yeast: 1. In the chromosome, I already integrated a DNA sequence (~800bp). What if I transform a replication plasmid with the same/very similar DNA sequence? Will the two recombine? 2. a DNA sequence (~1000bp) was integrated first into the chromosome. A second sequence, which has more than two domains of high similarity with the first integrated sequence. I wonder if recombination will happen. If so, will it have more than one crossover? Thanks, Jingjing From andrealr82 from hotmail.com Tue Jul 14 13:00:01 2009 From: andrealr82 from hotmail.com (=?iso-8859-1?Q?Andrea_Lo_R=E9?=) Date: Tue Jul 14 15:22:37 2009 Subject: [Yeast] asking for information Message-ID: Hello,Searching the net I found a response about liquid b-gal assays, and since you have vast experience on this issue I decided to write to you. I'm using pSV-Bgal for transfection normalization, so far I didn't get good results (at least not what I expected!), and I don't know if the problem is on the way used for detection. I'm doing assays with reporter systems and I must say I don't have much experience on this. I'm just starting my work (I'm a PhD student at Buenos Aires School of Medicine in Argentina), in fact I'm doing the first activity analysis in order to find the minimal promoter sequence of a gene. I did some in silico research to learn more about the promoter, and got some "potential" promoters (using Mat Inspector). My first attempt was to take a 2000bp fragment upstream exon 1 by PCR on HeLa cells and start activity measurements. The gene of interest codes for a protein that plays a role in autophagy, and its expression is upregulated in different stress situations included starvation. I cloned the sequence on pGL3 vector and checked activity after transfecting HeLa cells which were then treated under starvation conditions; as a control I used the pGL3 empty vector and for transfection control I used pSV40-Bgal. So far I didn't got good activity results......I don't find consistent differences between treated and untreated cells (we suppose that in normal conditions the promoter is not going to be very active, or better said.....under starvation conditions we expect to have a great activation of the expression, and therefore have a strong luciferase signal)...so after dealing a lot trying to find the reason....I now blame it on everything!!! I first considered that maybe 2000bp is not enough and I should go further upstream the ATG. Then I also wonder if the detection system is ok. I'm using a Firefly luc assay kit for pGL3 activity detection, and for Bgal I do a colorimetric measurement using ONPG at 420nm OD. I rinse the cells with passive buffer and use a sample for luminiscence and other for bgal. Every time I did it, samples took a long time to turn into yellow, and some of them even didn't changed at all, so results were so variable that I'm not sure about them..Do you have a standard protocol to measure B-gal activity? I also realised you normalized with protein concentration...In this case is it enough to have the b-gal value to normalize the luciferase results? or I should also take into account proteins concentration?I know you work with yeast but...= From Marija.Vujcic from roswellpark.org Tue Jul 21 14:02:21 2009 From: Marija.Vujcic from roswellpark.org (Vujcic, Marija) Date: Wed Jul 22 09:25:18 2009 Subject: [Yeast] yeast CGH analysis Message-ID: <6B3493ABEE194842A8EFC3FC3B23DE6A105BF41019@MSXMBCCR2.roswellpark.org> Dear colleagues, we are interested in doing yeast CGH and I have couple of questions: - did any of you use services of Nimblegen (Roche) for yeast CGH and how satisfied (or not) were you with the service/cost and what are your general comments? - do you know of any lab that successfully does yeast CGH and would be interested in collaborating with us (doing the CGH for us as a part of collaboration)? Thank you very much in advance for all your help, Marija Vujcic Roswell Park Cancer Institute Buffalo, NY This email message may contain legally privileged and/or confidential information. If you are not the intended recipient(s), or the employee or agent responsible for the delivery of this message to the intended recipient(s), you are hereby notified that any disclosure, copying, distribution, or use of this email message is prohibited. If you have received this message in error, please notify the sender immediately by e-mail and delete this email message from your computer. Thank you. -------------- next part -------------- An HTML attachment was scrubbed... URL: http://www.bio.net/bionet/mm/yeast/attachments/20090721/555b6c2a/attachment.html From jjsun from Princeton.EDU Fri Jul 24 14:25:01 2009 From: jjsun from Princeton.EDU (Jingjing Sun) Date: Sun Jul 26 15:39:44 2009 Subject: [Yeast] CUP1 promoter and Copper toxicity to S. cerevisae Message-ID: <4A6A0A8D.5080807@princeton.edu> Dear Yeast Netters, There is a question regarding CUP1 promoter: I placed a gene of interest under the CUP1 promoter. When the cupric sulphate is above 100 uM, I start to see the dark cell sediment on the tube bottom. I assume this is due to the copper toxicity. When the inducer goes up to 500 uM, I saw improved protein production, but the cell growth was inhibited a lot, and i saw debris by Flow cytometry. However, I checked with a paper ( Hottiger, T., Furst, P., Pohlig, G. & Heim, J. Physiological Characterization of the Yeast Metallothionein (Cup1) Promoter, and Consequences of Overexpressing Its Transcriptional Activator, Ace1. Yeast 10, 283-296 (1994)), the amount of cupric sulphate was much lower than the toxicity limit (2mM). Would you please share with me your experience on CUP1 promoter? Is there any substitute to replace CUP1 promoter if the toxicity is a really problem? Thanks a lot! Jingjing From alustig from tulane.edu Wed Jul 29 10:28:58 2009 From: alustig from tulane.edu (Arthur Lustig) Date: Wed Jul 29 10:30:59 2009 Subject: [Yeast] Postdoctoral Positions Message-ID: Dear Colleagues, I am searching for a postdoctoral fellow to work on telomere dynamics in yeast for a start date early in 2010. My major emphasis is on recombination between telomeres and the role of a regulatory region of the major repair and telomere protein, Mre11, in that process. We are also studying the mechanism of telomere rapid deletion as a model system for telomere size homeostasis. For a review of my interests, please see Lustig (2003) Nature Reviews Genetics, 4, 916. If interested, please contact me by e-mail or at the address below. Arthur J. Lustig PhD Professor, Department of Biochemistry Tulane University Health Sciences Center 1430 Tulane Avenue New Orleans, LA 70112 ph:504-988-3688 fax:504-988-3687 -------------- next part -------------- An HTML attachment was scrubbed... URL: http://www.bio.net/bionet/mm/yeast/attachments/20090729/6b5d53ce/attachment.html