From owner-recombination@net.bio.net Fri Apr 03 23:00:00 1998 Path: biosci!biosci!not-for-mail From: "G. Dellaire" Newsgroups: bionet.molbio.recombination Subject: Illegitimate Vs. Homologous recombination (yeast and mammals) Date: 4 Apr 1998 08:55:12 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 120 Sender: daemon@net.bio.net Approved: dellaire@odyssee.net Distribution: world Message-ID: <6g5olg$kar@net.bio.net> NNTP-Posting-Host: net.bio.net Hello, I have been working all day trying to work out the initial outline for my Ph.D. thesis. In any case, this work has lead me to confront an age old question. Why do yeast and mammalian cells differ so drastically with respect to the efficiency of illegitimate recombination and homologous/targeted recombination? As has been demonstrated over and over, yeast are extremely efficient at integrating DNA at homologous sites, whereas mammalian cells exhibit targeting frequencies that are lower by at least 2-3 orders of magnitude. In contrast, yeast are very inefficient at joining DNA ends that are non-homologous (i.e. illegitimate recombination) but mammals are extremely efficient at DNA end joining and hence illegitimate integration of DNA. I have been paging through the now "classic" _Genetic Recombination, (R. Kucherlapati and G.R. Smith Ed. (1988) ASM, Washington D.C.) and have read through the possible explanations for this discrepancy between yeast and mammals given by David Roth and John Wilson (Chapter 21: Illegitimate Recombination in Mammalian Cells, pg. 623-624). They give the following possibilities with associated caveats in parentheses: 1. Targeted recombination could be less efficient in mammalian cells because of the larger size of genome compared to yeast. The implication being, that more DNA must be "scanned" before homologous recombination can occur in mammals. (problem is that several studies indicate that homology search is not rate limiting (including my own data to be published) in mammalian cells. As well, organisms with the same genome size as S. cerevisiae (ex. S. pombe) do not show the same ratios of illegitimate recombination Vs. homologous recombination). Copy number is also invoked and then summarily debunked. In yeast higher copy number usually equals higher efficiency of targeting (ex. single loci vs. rDNA loci). In mammals the targeting at sites that occur multiple times (ex. ~200 times for ribosomal RNA genes and ~100,000 times for Line elements) is not more efficient than targeting at a single copy site. 2. Roth and Wilson conclude the enzymology may be different between yeast and mammalian recombination. Mammalian cells do seem to have an enhanced capacity for DNA end joining (perhaps as much so as Xenopus... which is notorious for this capacity) as compared to yeast. In addition, several genes implicated in mammalian DNA repair and recombination are not present (or no homologue has yet been found) in yeast. For example, S. cerevisiae lacks an ADP-polyribosylase and although not related per se to recombination directly (perhaps more to access of DNA), histone H1. So we are left to conclude that enzymology is a key part of solving this case of unequal frequencies. I would like to hear more comments on this but I have a more utilitarian question to ask. Why would it be beneficial for mammalian cells to develop a system of recombination (and associated enzymology) to carry out highly efficient illegitimate recombination perhaps at the detriment of homologous recombination. If you would all indulge me for the moment I will present my attempts to answer this question. 1. Genome Complexity -The mammalian genome contains many types of repetitive DNA (LINEs, SINEs, alpha DNA at centromeres etc.) as well as multigene families, all of which present many chances for homologous recombination between and within chromosomes. Mammalian genes are also disrupted by introns, increasing the amount of non-coding DNA which can withstand mutation without affecting a deleterious phenotype. Yeast, on the other hand, have more simplified genome with very few introns and repetitive elements. As well, yeast can exist as a haploid organism further reducing the levels of homology within its genome (i.e. no homologue to interact with). It is therefore my suggestion that mammalian cells have adapted a reduced capacity for homologous recombination in concert with the expansion of repetitive DNA families and tetraploidizations (to account for multigene families) that have occurred over the last billion years or so. If mammalian cells could still undergo homologous recombination at the same relative frequency as yeast, when compared to illegitimate recombination, the genome would rapidly be scrambled. Illegitimate recombination via rapid end joining of naturally occurring double strand breaks, in a complex genome, is a more easily survived event if you have a good chance that the end joining is occurring in non-coding DNA. In short, "Thank god for junk DNA". 2. Metazoans Vs. Protozoa -Yeast are unicellular, if only one member of a colony survives the organism can pass on its genes. Mammals being multicellular require the majority of their cells to maintain their genetic integrity, failure to do so leads to disease and ultimately the demise of the organism. Thus, when confronted with the same catastrophic event, a double strand break occurring in the organisms genome, yeast and mammalian cells will approach the repair of this break differently. Yeast can afford to have aberrant recombination leading to the death of a single yeast cell, as at least one of the colony will survive that cell. Mammals, in contrast, will repair that break as quickly as possible and that involves simple DNA end joining. By simply joining the ends of the DNA break, the mammalian cell has saved the integrity of its genome. Moreover, due to the complexity of its genome there is a good chance that the break occurred in non coding DNA. The yeast cell, on the other hand, may require efficient homologous recombination as most breaks will occur in coding DNA and the other homologue is the only intact template from which to copy any lost sequence. Comments? Hope to hear from somebody, hopefully someone who works in yeast or another non-mammalian system. P.J. Hastings? M. Lichten? S. Hawley? Cheers, Graham Dellaire Div. of Experimental Medicine McGill University dellaire@odyssee.net (also Moderator of RECOM) From owner-recombination@net.bio.net Mon Apr 06 23:00:00 1998 Path: biosci!biosci!not-for-mail From: Jeff Slater Newsgroups: bionet.molbio.recombination Subject: searchable science resources Date: 7 Apr 1998 07:41:43 -0700 Organization: BT Internet Lines: 9 Sender: daemon@net.bio.net Approved: dellaire@odyssee.net Distribution: world Message-ID: <6gddv7$evg@net.bio.net> NNTP-Posting-Host: net.bio.net I have located a free service for locating life science products at: http://www.corniche.com try it - there are on-line quote requests etc. Jeff Slater From owner-recombination@net.bio.net Thu Apr 16 23:00:00 1998 Path: biosci!biosci!not-for-mail From: maison Newsgroups: bionet.molbio.recombination Subject: Re: Queries on recombination events Date: 17 Apr 1998 15:15:43 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 42 Sender: daemon@net.bio.net Approved: dellaire@odyssee.net Distribution: world Message-ID: <6h8kaf$dco@net.bio.net> NNTP-Posting-Host: net.bio.net At 18:54 98-04-17 +0100, you wrote: Hello I came across your notes/messages posted on bionet on recombination and would be grateful if you could answer a couple of my queries . I am a Ph.D student in the Dept of Plant Sciences in Oxford. My queries are: a) What exactly is random site integration? What are it specific advantages and disadvantages. When a single copy gene ( or a transgene ) is randomly integrated , does it mean it is integrated at only ONE random site, if so then what are the chances of subsequent rearrangement? Also can such random integration and subsequent arrangement ever cause a 'variability' desirable or undesirable in the progeny . I am thinking more in terms of plant breeding (F2 generation) however all examples/ref from the non-plant sector will be of immense help. b) My next query is about random recombination events as opposed to site specific ( cre-lox etc) recombination. In the latter system what are the chances of fallibility or simply the transgene under the system, may by accident/chance is randomly integrated? Idid not come accross any message posted on these lines on the bionet recombination newsgroup. Probably I missed them....but I do hope you can help me . Many thanks KGhosh maison@sable.ox.ac.uk From owner-recombination@net.bio.net Thu Apr 16 23:00:00 1998 Path: biosci!biosci!not-for-mail From: Valeria Maida Newsgroups: bionet.molbio.recombination Subject: NAIMI Congress Date: 17 Apr 1998 06:58:21 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 68 Sender: daemon@net.bio.net Approved: dellaire@odyssee.net Distribution: world Message-ID: <6h7n5t$2f9@net.bio.net> NNTP-Posting-Host: net.bio.net It gives us great pleasure to inform you that the NAIMI Congress will be held in Alghero, on the North West of Sardinia, in the centre of Mediterranean Sea, this September as a satellite symposium to the Congress in Coordination Chemistry in Florence. This congress is specifically aimed at those working in the DNA Polymers field and promises to be a valuable and stimulating occasion. The opportunity for experts in the general chemical sector to meet those working on the base of DNA and RNA in an informal setting should lead to interesting brainstorming sessions, which will undoubtedly be of great use to both sides. Yours, Prof. M.L.Ganadu=A0 &=A0=A0 Prof. M. Taddei - Dipartimento di Chimica - via Vienna, 2 - 07100 Sassari=20 UNIVERSIT=C0 DEGLI STUDI DI SASSARI NAIMI Nucleic acids and their interactions with metal ions Satellite Symposium of the XXXIII International Conference on Coordination Chemistry in Florence Alghero September 5-7 1998 The mini symposium is intended for researchers in the Chemistry of Nucleic Acids and Metal Ions field. The main objective of the symposium is to bring together scientists interested in the different aspects of the influence of metal ions on the structure and metabolism of DNA. The main topics will be DNA Polymers, nucleic metal proteins, zinc fingers and PNA. Scientific Program: The following distinguished speakers have already agreed to deliver a lecture:=20 Prof. James Allan Cowan (COLUMBUS, USA),=A0=20 Prof. Rob Kaptein (UTRECHT, THE NETHERLANDS),=A0=A0=20 Prof. Bernard Meunier (CNRS, FRANCE),=A0=20 Prof. Thomas W. Myers (ROCHE, USA),=A0=A0=20 Prof. Huguette Pelletier (HOUSTON, USA).=A0=20 Prof. Bibudgendra Sarkar (TORONTO, CANADA),=A0=20 In addition there will be a poster session and a number of selected oral communications. All those who wish to participate are invited to submit an abstract to the scientific committee. Organising committee: F. Bonomi (University of Milano - Italy)=20 M.L. Ganadu, Chairperson (University of Sassari - Italy)=20 H. Kozlowski (University of Wrocklaw - Poland)=20 C. Mealli (CNR, Florence - Italy)=20 A. Scozzafava (University of Florence - Italy)=20 M. Taddei (University of Sassari - Italy)=20 additional information and a registration form is available on: http://www.uniss.it/web/congressi/naimi/naimiaw.htm For further information, please contact: Peter Norton peter.norton@flashnet.it=A0=A0=A0=A0=A0=A0=A0=A0=A0 phone/fax:= +39 79 299640 Naimi secretary naimi@ssmain.uniss.it=A0=A0=A0=A0=A0=A0=A0=A0=A0=A0=A0=A0=A0= =A0 fax +39 79 229559 or 229482 Newtours Evangelist@Mail.Newtours-CMO.it=A0=A0=A0=A0 Fax+3955/3361250/350 ********************************************************************* Valeria Maida Tel. + 39 79 229542/229588 =20 Dipartimento di Chimica fax + 39 79 229559/229482 =20 via Vienna, 2 e-mail billia@ssmain.uniss.it =20 07100 Sassari =20 ********************************************************************* =20 From owner-recombination@net.bio.net Fri Apr 17 23:00:00 1998 Path: biosci!biosci!not-for-mail From: BIOSCI Administrator Newsgroups: bionet.molbio.recombination Subject: BIOSCI/bionet miniFAQ & Fundraiser Date: 18 Apr 1998 05:50:45 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 234 Sender: daemon@net.bio.net Approved: dellaire@odyssee.net Distribution: world Message-ID: <6ha7j5$78n@net.bio.net> NNTP-Posting-Host: net.bio.net (LAST REVISION: 30-JUL-95) This BIOSCI "miniFAQ" is designed to answer the questions that come up the *most frequently*. 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Because of our limited personnel resources, we ask that you resubmit a *complete* form to revise your entry; we only replace complete entries and do not have resources to edit old forms. From owner-recombination@net.bio.net Fri Apr 17 23:00:00 1998 Path: biosci!biosci!not-for-mail From: Graham Dellaire Newsgroups: bionet.molbio.recombination Subject: Re:Illegitimate Vs. Homologous recombination (yeast and mammals) Date: 18 Apr 1998 06:54:00 -0700 Organization: McGill Div. of Experimental Medicine Lines: 130 Sender: daemon@net.bio.net Approved: dellaire@odyssee.net Distribution: world Message-ID: <6hab9o$e0r@net.bio.net> Reply-To: dellaire@odyssee.net NNTP-Posting-Host: net.bio.net A few people had e-mailed me with interest in Illegitimate Vs Homologous reombination in yeast and mammals. Although, no one came forward with their own ideas there seems to be enough interest that I thought I would post the section from my thesis pertaining to this issue. Comments are welcome, its not submitted yet and anymore ideas would be appreciated! Cheers, Graham Dellaire Moderator RECOM Mammalian cells are much more adept at illegitimate recombination than homologous recombination. The optimum reported frequencies of illegitimate integration of DNA, within the genome of a mammalian cell, range from 1 in 100 for microinjected DNA (Capecchi, 1980; Folger et al., 1982) to 1 in 1000 for transfected DNA (Adair et al. 1989; Merrihew et al., 1996). This is a global frequency for integration anywhere within the entire genome. Specific integration of DNA in mammalian cells, into a target locus through homologous recombination (i.e. gene targeting), occurs at frequencies of 1 in 1000 cells microinjected (Thomas et al., 1986; ) to 1 in 10e6-10e7 transfected cells (Song et al. 1987; Hasty et al., 1991a; Hasty et al., 1994). Thus, the ratio of homologous integration events versus the total number of illegitimate integration events is at best, 1 in 10 for microinjected DNA and 1 in 1000 for transfected DNA. This difference in recombination frequencies is a hallmark of mammalian cells. In contrast, the efficiency of homologous recombination in the yeast, S. cerevisiae, is at least 10 fold greater than illegitimate recombination in this organism (Hinnen et al., 1978; Scherer and Davis, 1979; Rothstein, 1983). Enzymology of recombination may be different between yeast and mammalian somatic cells The differences in the relative frequencies of homologous versus illegitimate recombination in mammals compared to yeast, may be explained by differences in the enzymology of recombination between these organisms. Homologues of two genes implicated in DNA repair in mammalian cells, poly (ADP-ribose) polymerase (PARP) (Cleaver and Morgan, 1991; Chatterjee and Berger, 1994) and BRCA1 (Scully et al., 1997), have not been found in S. cerevisiae. It has been hypothesized, that PARP may be involved in minimizing recombination between repetitive elements in mammalian cells and one reason yeast exhibit such high rates of homologous recombination, even in the absence of repetitive elements, may be due to the absence of PARP in this organism (Lindahl et al., 1995; Jeggo, 1998). In agreement with this hypothesis, inhibition of PARP leads to an increase in spontaneous intrachromosomal homologous recombination (Waldman and Waldman, 1991). Another protein that may be responsible, in part, for the avoidance of illegitimate end-joining in yeast, is RAD5. Rad5 deletion mutants exhibit elevated levels of illegitimate recombination and show similar end repair junctions to those seen in mammalian cells (Ahne et al., 1997). In addition, homologous or illegitimate recombination can be selectively inhibited in yeast by mutation of either rad52 or rad50, respectively (Schiestl et al., 1994). For example, deletion of Rad50 (similarly for XRS2 or MRE11) reduces illegitimate recombination in yeast by up to 70 fold (Moore and Haber, 1996). Further support for the idea that differences in the enzymology of recombination may alter the ratio of illegitimate vs. homologous recombination, has come from the study of a chicken transformed B-cell line, DT40. The DT40 cell line can undergo homologous recombination and gene targeting at frequencies that are one to two orders of magnitude greater than seen in non B cell lines derived from the chicken (Buerstedde and Takeda, 1991). When the ratio of inter to intrachromosomal recombination events is measured in mammalian mitotic cells, the ratio is 1500 fold less than in spermatids (meiotic cells) and 500 fold less than for mitotic cells in yeast (Shulman et al., 1995). Ectopic homologous recombination between non allelic sequences and repetitive elements is elevated in mammalian germ line cells (Murti et al., 1994; Pittman and Schimenti, 1998) exhibiting a similar ratio of inter to intrachromosomal frequencies as seen in meiotic yeast cells (Shulman et al., 1995). It has been recently shown, that when human chromosomal sequences are cloned in yeast, a similar distribution of crossover sites occur within the cloned DNA as seen during human in vivo meiotic recombination (Campbell et al., 1995). This data would suggest, that rather than comparing recombination in yeast with that of mammalian somatic cells which do not readily undergo homologous recombination, it is more suitable to compare mammalian meiotic recombination to that of yeast. Of note, several mammalian proteins implicated in meiotic recombination that have homologues in yeast, including ATM (MEC1 in yeast, Brush et al., 1996; Keegan et al., 1996), RPA (Gailus-Durner et al., 1997; Plug et al., 1997) and RAD51 (Bishop 1994; Haaf et al., 1995), are found to be expressed at elevated levels in mammalian germline tissue (Shinohara et al., 1993; Chen and Lee, 1996). Efficient illegitimate recombination may be tolerated by mammalian cells due to the complexity of the mammalian genome. Mammalian genomes contain many repetitive elements (LINEs, Martin, 1990; SINEs, Okada, 1991; alphoid satellite, Musich et al., 1980; and centromeric repeats, Lee et al., 1997), their genes are disrupted by introns and the overall amount of DNA is several times that of yeast. In contrast, the genome of S. cerevisiae has fewer repetitive elements and most of the non-ribosomal genes lack introns (Rodriguez-Medina and Rymond, 1994). Typically, tracts of homology greater than a few kilobases are required for efficient homologous recombination in mammalian cells (Deng and Capecchi, 1992). Yeast in contrast require much shorter tracks of homology, in the range of 70-90 base pairs (Sugawara and Haber, 1992). The requirement of larger tracts of homology in mammalian cells strongly disfavors recombination between short blocks of homology. Since typically, most repetitive elements with the mammalian genome are less than 1 kb, the requirement for extended regions of homology may have developed as a mechanism for preventing recombination between repetitive elements (Deng and Capecchi, 1992). Double strand breaks (DSBs) are generated during normal cellular processes such as DNA transcription and replication (Roth and Wilson, 1988). When breaks do occur, illegitimate recombination provides a means for rapidly joining free DNA ends and thus maintaining the integrity of the cells genome. Loss of Ku antigen end binding activity, which affects DNA end joining directly (Ramsden and Gellert, 1998), can lead to enhanced sensitivity to ionizing radiation and genome instability (Getts and Stomato, 1994). The chance a DSB will occur in non coding DNA is much more likely in a complex genome containing introns and repetitive elements than a simple one. Breaks that occur in non coding DNA when repaired imprecisely will produce silent mutations. Multicellular organisms can also tolerate imprecise or aberrant recombination because most of the cells in these organisms are somatic and genetic changes in these cells are not heritable (Roth and Wilson, 1988). Copyright Graham Dellaire 1998 Unauthorized use or distribution of this material is prohibited. From owner-recombination@net.bio.net Fri Apr 17 23:00:00 1998 Path: biosci!biosci!not-for-mail From: Graham Dellaire Newsgroups: bionet.molbio.recombination Subject: Re: Re: Queries on recombination events Date: 18 Apr 1998 06:43:27 -0700 Organization: McGill Div. of Experimental Medicine Lines: 98 Sender: daemon@net.bio.net Approved: dellaire@odyssee.net Distribution: world Message-ID: <6haalv$bir@net.bio.net> Reply-To: dellaire@odyssee.net NNTP-Posting-Host: net.bio.net You Wrote: >My queries are: >a) What exactly is random site integration? The notion of random integration of plasmid DNA into the genome came from early studies where the illegitimate integration of plasmid DNA was monitored by Southern analysis and each clone appeared to integrate DNA at different sites in the genome (Pellicer et al., 1978; Folger et al., 1982). This was not however direct evidence. Only in the last few years has direct evidence of random integration been presented by the rescue of DNA sequences from gene trap vectors in murine embryonic stem cells (Chowdhury et al., 1997) and by FISH analysis of transgene integration at mega base resolution in murine fibroblasts (Dellaire and Chartrand, 1998). Challenges to the notion of random integration have come from many corners. There are often hotspots for meiotic recombination in both yeast and mammals (Lichten and Goldman, 1995), many illegitimate events involve microhomologies (Roth and Wilson, 1988 in Genetic Recombination, ASM Press), and Merrihew et al. (1996) has shown that previous integration sites in CHO cells can integrate DNA at higher rates than expected for random integration. Integration sites may be site of particular DNA structure as often (9/10) integration sites of both retro virus and plasmid DNA contain bent DNA elements (Milot et. al., 1992 and 1994). As well fragile sites in mammalian cells are also frequent sites of transgene integration in CHO cells (Rasool et al., 1991, 1992). In my opinion, on the local scale of a a few 10's of kb you may see differences in recombination. Over the entire scale of the genome at megabase resolution, microhomologies, fragile sites and bent DNA may occur often enought that for all intents and purposes integration of plasmid DNA is random. >What are it specific >advantages and disadvantages. When a single copy gene ( or a transgene >) >is randomly integrated , THe advantage of single site integration are that 1) Southern analysis and analysis of any rearrangements are simplified; 2)you won't have to worry about multicopy transgenes repression of your the gene in your plasmid; and 3)It makes it much more simple to rescue the DNA afterwards if you want to analyse the junctions or find out where you sequence is. >does it mean it is integrated at only ONE >random site, if so then what are the chances of subsequent >rearrangement? Also can such random integration and subsequent >arrangement ever cause a 'variability' desirable or undesirable in the >progeny . My laboratory (Dellaire et al, 1997; Dellaire and Chartrand, 1998) has shown directly that >90% of the time transgene integration occurs at a single site even when multiple copies are present. Capecchi and collegues has shown indirectly by Southern that single site insertion occurs for multicopy transgenes (Folger et al., 1992) Rearrangement can occur often during the integration process and we have direct evidence by FISH that in a population, transgenes can rearrange after integration in a minority of cells (manuscript in preparation). Depending on the organism and structure of a multicopy insertion (ex. how many repeated genes) you may get unequal cross over occuring and you may gain or lose repeats. This may effect the level of transgene expression. I think you will have to do the experiment and try to do single copy integrations by serial dilution of plasmid DNA used in your transfection. YOu can also use Cre-Lox or Flp-frt systems to cut out all copies leaving only one copy at the insertion site. References in plants: Look for work by H. Puchta and M.E. Offringa. >I am thinking more in terms of plant breeding (F2 generation) >however all examples/ref from the non-plant sector will be of immense >help. >b) My next query is about random recombination events as opposed to >site >specific ( cre-lox etc) recombination. In the latter system what are >the >chances of fallibility or simply the transgene under the system, may by >accident/chance is randomly integrated? I would have to look at the efficiencies but illegitimate integration occurs at rate of about 1 in 1000 cells transfected. If site directed recombination is more efficient than this then you would expect to see more specific integrations than "random". Maybe someone on the thread could give us an idea of efficiencies. I know that you can increase gene targeting by 6-20 fold using CRe-Lox(Merrihew et al. (1995)). I would assume that simple integration of DNA would be much higher though. I would appreciate it if others would lend there thoughts, especially those of you out there who work in plants and/or use cre-lox systems. Cheers, Graham Dellaire Div. Exp. Medicine McGill University dellaire@odyssee.net From owner-recombination@net.bio.net Sun Apr 19 23:00:00 1998 Path: biosci!biosci!not-for-mail From: Graham Dellaire Newsgroups: bionet.molbio.recombination Subject: Job: apillomavirus DNA Replication and Genome Segregation Date: 19 Apr 1998 20:46:27 -0700 Organization: McGill Div. of Experimental Medicine Lines: 30 Sender: daemon@net.bio.net Approved: dellaire@odyssee.net Distribution: world Message-ID: <6hegej$mal@net.bio.net> Reply-To: dellaire@odyssee.net NNTP-Posting-Host: net.bio.net From: alison_mcbride@nih.gov Subject: Papillomavirus DNA Replication and Genome Segregation Papillomavirus DNA Replication and Genome Segregation Alison A. McBride, PhD Papillomavirus DNA Replication and Genome Segregation Alison McBride, PhD Papillomaviruses replicate and maintain their genomes as extrachromosomal elements within infected cells. A postdoctoral position is available to study the mechanisms by which these viruses maintain and segregate their genomes. Candidates should have a strong background in molecular biology, biochemistry or cell biology. Experience with in situ hybridization would be helpful. Applicants must have a PhD and/or MD and less than five years of postdoctoral experience. Laboratory of Viral Diseases, NIAID, Building 4, room 137, 4 CENTER DR MSC 0455,Bethesda, MD 20892-0455 Voice: 301-496-1370 Fax: 301-480-1497. To request further information about this opportunity, or apply it online, visit this location on the NIH World Wide Web site: http://helix.nih.gov:8001/oe/laboratory/main.phtml?key=870899701 This is just a one of several current opportunities for postdoctoral training at the NIH. We also have openings for physicians in our clinical training programs and openings for tenure-track investigators. Check out the details, request further information and apply online today at the NIH Web site. From owner-recombination@net.bio.net Sun Apr 19 23:00:00 1998 Path: biosci!biosci!not-for-mail From: Graham Dellaire Newsgroups: bionet.molbio.recombination Subject: Gene Therapy 2 post-Docs Date: 19 Apr 1998 20:46:00 -0700 Organization: McGill Div. of Experimental Medicine Lines: 31 Sender: daemon@net.bio.net Approved: dellaire@odyssee.net Distribution: world Message-ID: <6hegdo$m7s@net.bio.net> Reply-To: dellaire@odyssee.net NNTP-Posting-Host: net.bio.net From: wj2411@medzilla.com (MEDZILLA-JOBS) Subject: Postdoctoral position in gene therapy Date: 15 Apr 1998 01:29:47 GMT An opening is available for one or two postdoctoral fellows in the design and construction of viral vectors for gene delivery. Targets for gene delivery using this approach involve genetic and acquired immune deficiencies (including AIDS), malignant tumors and diseases involving excessive inflammation that could be limited by delivery of anti- inflammatory cytokines and enzymes to a target organ. The individual is given considerable responsiblity in planning and executing his/her experiments, and must have experience in basic molecular techniques: cloning; sequencing DNA; Northern, Southern and Western analyses; plasmid construction and PCR; gene expression technology; and cell culture. Work with small animals (mice, rats, rabbits) required. Prior postdoctoral experience desirable but not necessary. Prior experience in gene therapy field not necessary. Salary commensurate with experience. Potential of advancement to junior faculty level position if productive. Send CV, and a list of a minimum of 3-4 references. E-mail application and additional details: http://www.medzilla.com/jobs/wj2411.htm From owner-recombination@net.bio.net Sun Apr 19 23:00:00 1998 Path: biosci!biosci!not-for-mail From: "G. Dellaire" Newsgroups: bionet.molbio.recombination Subject: 4 year appointment funded by the Wellcome Trust! Date: 20 Apr 1998 05:56:33 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 40 Sender: daemon@net.bio.net Approved: dellaire@odyssee.net Distribution: world Message-ID: <6hfgm1$lmf@net.bio.net> NNTP-Posting-Host: net.bio.net From: t.j.keen@leeds.ac.uk (T. Jeffrey Keen) Subject: Postdoctoral position Date: Tue, 3 Mar 1998 12:11:10 +0000 (GMT) Molecular Medicine Unit, St James's Hospital, Leeds Postdoctoral Research Fellow St James's University Hospital is the largest teaching hospital in Europe, and a leading centre for medical research in the UK. The Molecular Medicine Unit has diverse interests including inherited defects of vision and hearing, cancer genetics, complex trait mapping, virology and gene therapy. Retinitis pigmentosa is a human inherited retinal degeneration which is a major cause of blindness in the under 65 age group. We are looking for an enthusiastic researcher to join an active group working on this and other forms of human inherited blindness. The group have succesfully identified three loci for dominant retinitis pigmentosa (Nat Genet 4;51-53, Hum Mol Genet 3;351-354, Hum Mol Genet 4:1459-1462) in collaboration with colleagues at Moorfields Eye hospital and the Institute of Ophthalmology, London. The successful applicant will use the techniques of linkage analysis and mutation detection to look for new proteins involved in RP, then study them further in order to better understand how the normal eye functions, as well as how mutations can lead to blindness. This is a four year appointment funded by the Wellcome Trust, at a point on the Trust's enhanced salary scale dependant on age and experience, and includes laboratory running costs and travel funds to attend international conferances. Applicants must already have a PhD in a relevant dicipline and a track record of research in a similar field would be an advantage. Applications with CV to Dr Chris Inglehearn at the above address by 27th March, for a start date asap thereafter. For informal enquiries phone 0113 206 5698 or Email cinglehe@hgmp.mrc.ac.uk. From owner-recombination@net.bio.net Mon Apr 20 23:00:00 1998 Path: biosci!biosci!not-for-mail From: "Dietmar Winkler" Newsgroups: bionet.molbio.recombination Subject: problem with terminology (ALU REPEAT?) Date: 21 Apr 1998 13:06:53 -0700 Organization: Vienna University, Austria Lines: 18 Sender: daemon@net.bio.net Approved: dellaire@odyssee.net Distribution: world Message-ID: <6hiu8t$r7r@net.bio.net> NNTP-Posting-Host: net.bio.net Hello! Does anybody know the meaning of the term "ALU REPEAT SEQUENCE" ? All help on this would be greatly appreciated! Thanks in advance, Dietmar. From owner-recombination@net.bio.net Mon Apr 20 23:00:00 1998 Path: biosci!biosci!not-for-mail From: toukie@zui.unizh.ch (Dr. S. Shapiro) Newsgroups: bionet.molbio.recombination Subject: Criticisms of Ames mutagencitiy tests Date: 21 Apr 1998 05:40:14 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 18 Sender: daemon@net.bio.net Approved: dellaire@odyssee.net Distribution: world Message-ID: <6hi43e$jtp@net.bio.net> NNTP-Posting-Host: net.bio.net Dear Colleagues; I am seeking original articles or reviews describing problems with the Ames mutagenicity test for prediction of mutagenic or carcinogenic activities of chemicals towards mammalian organisms (particularly though not exclusively humans or human cell lines). I would appreciate receiving complete citations (author, journal, volume, page, year) so that I may check out these articles myself. Kindly respond to me _directly_ at toukie@zui.unizh.ch Thanks in advance to all responders, S. Shapiro toukie@zui.unizh.ch From owner-recombination@net.bio.net Mon Apr 20 23:00:00 1998 Path: biosci!biosci!not-for-mail From: "Tarran Jones" Newsgroups: bionet.molbio.recombination Subject: Postdoc in Antibody Engineering Date: 21 Apr 1998 05:42:43 -0700 Organization: MEDICAL RESEARCH COUNCIL Lines: 42 Sender: daemon@net.bio.net Approved: dellaire@odyssee.net Distribution: world Message-ID: <6hi483$k47@net.bio.net> NNTP-Posting-Host: net.bio.net Antibody Engineering Group, MRC Collaborative Centre, 1-3 Burtonhole Lane, Mill Hill, London, NW7 1AD. UK.=20 The MRC Collaborative Centre is affiliated to the Medical Research Council and acts as a commercial interface between the MRC's research base and industry. Within the Centre, the Antibody Engineering Group works directly with both pharmaceutical and biotechnology companies on a variety of antibody-based collaborative research projects and has a World-wide reputation for successfully humanising antibodies. As part of our continuing expansion, the group is now looking for an experienced and motivated=20 POSTDOCTORAL SCIENTIST The successful applicant will take on increasing responsibility for running research projects and interacting with our collaborative partners. Moreover, the opportunity exists to develop ideas on how antibody engineering may be used to create the next generation of therapeutic agents. Practical experience in recombinant DNA technology and the ability to work as part of a team are essential. Previous experience of antibody engineering and/or the expression of recombinant proteins would be an advantage.=20 ------------------------------------------------------------------------ The MRC Collaborative Centre operates a unified pay and grading system along with an optional pension scheme. Salaries will be in the range =A320.094 - =A329,710 per annum and appointments will be for 2 years in the first instance with the possibility of extension.=20 Applicants should send a curriculum vitae including the names of two referees and details of current level of remuneration to Mrs Sandra Gibney at the above address. Please quote reference CC/ABE3/PS. Informal enquiries may be made to Dr. Tarran Jones by Phone: +44 (0)181 906 3811, Fax: +44 (0)181 906 1395, or E-mail: t-jones@nimr.mrc.ac.uk. The closing date for the receipt of applications will be Monday, 18th May, 1998.=20 The MRC Collaborative Centre is an Equal Opportunities Employer and actively discourages smoking. From owner-recombination@net.bio.net Tue Apr 21 23:00:00 1998 Path: biosci!biosci!not-for-mail From: Bob Lansman Newsgroups: bionet.molbio.recombination Subject: Definition of ALU repeat Date: 22 Apr 1998 14:39:50 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 28 Sender: daemon@net.bio.net Approved: dellaire@odyssee.net Distribution: world Message-ID: <6hlo36$des@net.bio.net> NNTP-Posting-Host: net.bio.net The alu repeats are a sequence which is repeated more than 700,000 times in the genomes of humans and other primates. The sequence is called Alu because most copies, (about 300 bp long) have a single cleavage site for the restriction endonuclease Alu. They are widely scattered throughout the genome between genes and in introns. They can transpose through RNA intermediates, causing insertional mutations. In this respect they are typical of the "SINES" (short, interspersed repeats) which have been found in all vertebrate genomes. They have no known function in the genome and are commonly thought-of as junk or selfish DNA. While they clearly do reduce the fitness of individual organisms slightly, they are widely thought to increase the evolutionary potential of the species genome perhaps by serving as "portable units of homology". Illegitimate recombination between non-allelic Alus could cause gene duplication and deletion, inversions, translocations and other chromosomal mutations of the type which could produce novelty. I'd like to see a thread started considering the possibilities of illegitimate recombination and SINES. It is my intuitive sense that many aspects of the structure of the vertebrate genome reduce the fitness of individuals but greatly enhance evolvability. Comments? Bob <<<<<<<<<<< >>>>>>>>>> If we make evolution illegal, only outlaws will evolve. Bob Lansman, Dept. of Biology and Microbiology University of Wisconsin Oshkosh Oshkosh, WI 54901 (414) 424-7089 FAX (414) 424-1101 From owner-recombination@net.bio.net Tue Apr 21 23:00:00 1998 Path: biosci!biosci!not-for-mail From: "David.Humair@bota.unine.ch"@NEDCU4.UNINE.CH Newsgroups: bionet.molbio.recombination Subject: BY2 transformation Date: 22 Apr 1998 05:46:55 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 33 Sender: daemon@net.bio.net Approved: dellaire@odyssee.net Distribution: world Message-ID: <6hkorv$t31@net.bio.net> NNTP-Posting-Host: net.bio.net Relay-Version: ANU News - V6.1B10 04/18/95 OpenVMS AXP; site news Path: news!nntp Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins, Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins, bionet.molbio.proteins.fluorescent,bionet.molbio.recombination,bionet.plants ,bionet.plant.signaltransduc Subject: BY2 transformation Message-ID: <353DE019.7B25A990@bota.unine.ch> From: David Humair Date: Wed, 22 Apr 1998 14:18:35 +0200 Reply-To: David.Humair@bota.unine.ch Organization: Labo de biochimie Universit=E9 de Neuch=E2tel Nntp-Posting-Host: mac20-24.unine.ch X-Mailer: Mozilla 4.04 (Macintosh; I; PPC) MIME-Version: 1.0 Content-Type: text/plain; charset=3Dus-ascii; x-mac-type=3D"54455854"; x-mac-reator=3D"4D4F5353" Content-Transfer-Encoding: 7bit Lines: 10 Hi netters, I'm trying to transform BY2 cells with the PEG method. It doesn't work. In fact, I'm trying to apply the PEG transformation protocol that we use for normal tobacco cells protoplasts. Does somebody have a good protocol to transform BY2 protoplasts with PEG? Thank you very much, David Humair From owner-recombination@net.bio.net Tue Apr 28 23:00:00 1998 Path: biosci!biosci!not-for-mail From: Graham Dellaire Newsgroups: bionet.molbio.recombination Subject: Gene Therapy JOBS from Bionet.employment Date: 28 Apr 1998 19:38:48 -0700 Organization: McGill Div. of Experimental Medicine Lines: 127 Sender: daemon@net.bio.net Approved: dellaire@odyssee.net Distribution: world Message-ID: <6i63ro$afj@net.bio.net> Reply-To: dellaire@odyssee.net NNTP-Posting-Host: net.bio.net The following is a list of recent job positions in Gene Therapy advertised in EMPLOYMENT/bionet.jobs.offered http://www.bio.net/hypermail/EMPLOYMENT/ ------------------------------------------- From: wj2426@medzilla.com (MEDZILLA-JOBS) Subject: Postdoctoral Positions in Molecular Biology and Gene Therapy Research Jefferson Medical College Date: 28 Apr 1998 00:26:01 GMT NIH-funded postdoctoral research positions are available for individuals with experience in molecular biology who are interested in cellular signaling as it regulates type II alveolar cell secretory functions (1), virus-cell interactions as they regulate cellular gene expression and cell death (1) and virus-mediated gene transfer in vector development for gene therapy (1). Experience working with cell culture and nucleic acids is necessary: cloning, DNA and RNA isolation, manipulation and analysis. Expertise with protein analysis, SDS-PAGE, and Western blotting is desirable. Applicants must be either citizens or Permanent Residents of the U.S.A. Contact: David S. Strayer, MD, PhD, Department of Pathology and Cell Biology, Jefferson Medical College, 1020 Locust Street, Philadelphia, PA 19107. Response by () or fax (215-923-2218) is encouraged. Start dates, salaries, etc. negotiable. Please supply C.V., contact phone numbers references + telephone numbers. References: virus-cell interactions Wali, A., and Strayer, D.S.: Regulation of p53 gene expression by a poxviral transcription factor. Virology, 224:63-72(1996). cellular signaling Strayer, D.S., Korutla, L., and Thomas, A.P.: Surfactant protein-A receptor-mediated inhibition of calcium signaling in alveolar type II cells. Receptors & Signal Transd., 7:111-120(1997). virus-delivery for genetic therapy Strayer, D.S., Kondo, R., Milano, J., and Duan, L.-X.: Use of SV40-based vectors to transduce foreign genes to normal human peripheral blood mononuclear cells. Gene Therapy, 4:219-225(1997). E-mail application and additional details: http://www.medzilla.com/jobs/wj2426.htm --------------------------------------------- From: wj2411@medzilla.com (MEDZILLA-JOBS) Subject: Postdoctoral position in gene therapy Date: 28 Apr 1998 00:23:44 GMT An opening is available for one or two postdoctoral fellows in the design and construction of viral vectors for gene delivery. Targets for gene delivery using this approach involve genetic and acquired immune deficiencies (including AIDS), malignant tumors and diseases involving excessive inflammation that could be limited by delivery of anti- inflammatory cytokines and enzymes to a target organ. The individual is given considerable responsiblity in planning and executing his/her experiments, and must have experience in basic molecular techniques: cloning; sequencing DNA; Northern, Southern and Western analyses; plasmid construction and PCR; gene expression technology; and cell culture. Work with small animals (mice, rats, rabbits) required. Prior postdoctoral experience desirable but not necessary. Prior experience in gene therapy field not necessary. Salary commensurate with experience. Potential of advancement to junior faculty level position if productive. Send CV, and a list of a minimum of 3-4 references. E-mail application and additional details: http://www.medzilla.com/jobs/wj2411.htm ------------------------------------------------- From: jreiser@helix.nih.gov Subject: Gene Therapy Gene Therapy Jakob Reiser, PhD Two (2) positions are available in July 1998 to develop and test new viral vectors for use in gene therapy. Vectors based on lentiviruses are currently under investigation. These vectors will be improved using evolutionary strategies and subsequently tested in animal models. Methods are being developed for delivering genes to the brain. Highly motivated applicants with less than five years of postdoctoral experience should apply. DMNB, NINDS, Building 10, Room 3D04, 10 CENTER DR MSC 1260, BETHESDA MD 20892-1260. To request further information about this opportunity, or apply it online, visit this location on the NIH World Wide Web site: http://helix.nih.gov:8001/oe/laboratory/main.phtml?key=870899701 This is just a one of several current opportunities for postdoctoral training at the NIH. We also have openings for physicians in our clinical training programs and openings for tenure-track investigators. Check out the details, request further information and apply online today at the NIH Web site. From owner-recombination@net.bio.net Thu Apr 30 23:00:00 1998 Path: biosci!biosci!not-for-mail From: Graham Dellaire Newsgroups: bionet.molbio.recombination Subject: Gene Therapy JOBS from Bionet.employment Date: 1 May 1998 09:34:28 -0700 Organization: McGill Div. of Experimental Medicine Lines: 127 Sender: daemon@net.bio.net Approved: dellaire@odyssee.net Distribution: world Message-ID: <6ictik$31b@net.bio.net> Reply-To: dellaire@odyssee.net NNTP-Posting-Host: net.bio.net The following is a list of recent job positions in Gene Therapy advertised in EMPLOYMENT/bionet.jobs.offered http://www.bio.net/hypermail/EMPLOYMENT/ ------------------------------------------- From: wj2426@medzilla.com (MEDZILLA-JOBS) Subject: Postdoctoral Positions in Molecular Biology and Gene Therapy Research Jefferson Medical College Date: 28 Apr 1998 00:26:01 GMT NIH-funded postdoctoral research positions are available for individuals with experience in molecular biology who are interested in cellular signaling as it regulates type II alveolar cell secretory functions (1), virus-cell interactions as they regulate cellular gene expression and cell death (1) and virus-mediated gene transfer in vector development for gene therapy (1). Experience working with cell culture and nucleic acids is necessary: cloning, DNA and RNA isolation, manipulation and analysis. Expertise with protein analysis, SDS-PAGE, and Western blotting is desirable. Applicants must be either citizens or Permanent Residents of the U.S.A. Contact: David S. Strayer, MD, PhD, Department of Pathology and Cell Biology, Jefferson Medical College, 1020 Locust Street, Philadelphia, PA 19107. Response by () or fax (215-923-2218) is encouraged. Start dates, salaries, etc. negotiable. Please supply C.V., contact phone numbers references + telephone numbers. References: virus-cell interactions Wali, A., and Strayer, D.S.: Regulation of p53 gene expression by a poxviral transcription factor. Virology, 224:63-72(1996). cellular signaling Strayer, D.S., Korutla, L., and Thomas, A.P.: Surfactant protein-A receptor-mediated inhibition of calcium signaling in alveolar type II cells. Receptors & Signal Transd., 7:111-120(1997). virus-delivery for genetic therapy Strayer, D.S., Kondo, R., Milano, J., and Duan, L.-X.: Use of SV40-based vectors to transduce foreign genes to normal human peripheral blood mononuclear cells. Gene Therapy, 4:219-225(1997). E-mail application and additional details: http://www.medzilla.com/jobs/wj2426.htm --------------------------------------------- From: wj2411@medzilla.com (MEDZILLA-JOBS) Subject: Postdoctoral position in gene therapy Date: 28 Apr 1998 00:23:44 GMT An opening is available for one or two postdoctoral fellows in the design and construction of viral vectors for gene delivery. Targets for gene delivery using this approach involve genetic and acquired immune deficiencies (including AIDS), malignant tumors and diseases involving excessive inflammation that could be limited by delivery of anti- inflammatory cytokines and enzymes to a target organ. The individual is given considerable responsiblity in planning and executing his/her experiments, and must have experience in basic molecular techniques: cloning; sequencing DNA; Northern, Southern and Western analyses; plasmid construction and PCR; gene expression technology; and cell culture. Work with small animals (mice, rats, rabbits) required. Prior postdoctoral experience desirable but not necessary. Prior experience in gene therapy field not necessary. Salary commensurate with experience. Potential of advancement to junior faculty level position if productive. Send CV, and a list of a minimum of 3-4 references. E-mail application and additional details: http://www.medzilla.com/jobs/wj2411.htm ------------------------------------------------- From: jreiser@helix.nih.gov Subject: Gene Therapy Gene Therapy Jakob Reiser, PhD Two (2) positions are available in July 1998 to develop and test new viral vectors for use in gene therapy. Vectors based on lentiviruses are currently under investigation. These vectors will be improved using evolutionary strategies and subsequently tested in animal models. Methods are being developed for delivering genes to the brain. Highly motivated applicants with less than five years of postdoctoral experience should apply. DMNB, NINDS, Building 10, Room 3D04, 10 CENTER DR MSC 1260, BETHESDA MD 20892-1260. To request further information about this opportunity, or apply it online, visit this location on the NIH World Wide Web site: http://helix.nih.gov:8001/oe/laboratory/main.phtml?key=870899701 This is just a one of several current opportunities for postdoctoral training at the NIH. We also have openings for physicians in our clinical training programs and openings for tenure-track investigators. Check out the details, request further information and apply online today at the NIH Web site.