bchso@uhnix2.UUCP (Dan Davison) (05/23/87)
In article <1991@husc6.UUCP> gallagher@husc4.UUCP (paul gallagher) writes: >Prokaryotes exchange genetic information >with plasmids... And via viruses. Bacillus sp. also pick up DNA directly from the environment, ie from those around them that have died and lysed the cell membrane. >I think it is believed that the Progenotes - the ancestral cells - gave rise >to the Archebacteria, Eubacteria, and the Urkaryotes (a completely extinct >kingdom). I'm currently working with George Fox, the co-discoverer of the archebacterial kingdom, and although I don't have my latest Carl Woese paper handy I'm pretty sure the Urkaryotes didn't die out...we is them, or they is us, so to speak. >The mitochondria and perhaps the nucleus of the eukaryotic cell >are thus the descendants of eubacteria, and the cytoplasm of the cell is the >descendant of urkaryotes. This is the distinction as I understand it. The three kingdoms (not four) branch *very* deeply and fairly close to one another, although there are some very strange eukaryotes now being examined. >Later, another eubacterium containg chlorophyll >started living inside the cell, and this became the chloroplast of >photosynthetic protists and plants. I am studying the small subunit ribosomal RNA, the 16S rRNA (BTW, "S" is not a unit of length, it's a hydrodynamic measurement more roughly proportional to shape and *size*. 12S vs. 16S is meaningful when comparing two RNAs, but not when comparing an RNA and a protein-RNA complex like the ribosome (70S in eubacterial and archebacteria, 80S in most eucaryotes). The secondary structure of that RNA (the way the chain folds back on itself) is remarkably like the gram-negative eubacteria. If you did not know it was a chloroplast you'd put it in with the gram-negatives. > Paul Gallagher dr. dan davison/ Dept of Biochemical and Biophysical Sciences/ U. of Houston bitnet: bchs6\@uhupvm1.bitnet | 4800 Calhoun/ Houston, Tx 77004 arpanet: davison\@sumex-aim.stanford.edu|uucp:...rice!soma!uhnix1!uhnix2!bchsup
eddy@boulder.Colorado.EDU (Sean Eddy) (05/30/87)
In article <382@uhnix2.UUCP> bchso@uhnix2.UUCP (Dan Davison) writes: >In article <1991@husc6.UUCP> gallagher@husc4.UUCP (paul gallagher) writes: >>Prokaryotes exchange genetic information >>with plasmids... > >And via viruses. Bacillus sp. also pick up DNA directly from the environment, >ie from those around them that have died and lysed the cell membrane. Dan, is it true that only the Bacillus species can pick up and incorporate DNA from the environment? I was under the possibly naive impression that pretty much all the bacteria could do this trick, called transformation. The principle of bacterial transformation is the heart of genetic engineering in E. coli (admittedly, coli transformation is artificially enhanced in the laboratory), as well as the crucial point in the classical experiments of Avery, McCleod, and McCarty that indicated DNA is the genetic material (incidentally, their experiments used Diplococcus). I'll be interested to hear if my concept of transformation is indeed overly naive. - Sean Eddy - Dept. of Molecular, Cellular, Developmental Biology - Univ. of Colorado, Boulder; Boulder, CO 80309 - - "It's hard to argue with someone who knows what he's talking about." - - Craig Werner (MD/PhD '91) - "Everything you know is wrong." - - Firesign Theatre
werner@aecom.UUCP (06/02/87)
> Dan, is it true that only the Bacillus species can pick up > and incorporate DNA from the environment? I was under the possibly > naive impression that pretty much all the bacteria could do this > trick, called transformation. The principle of bacterial > transformation is the heart of genetic engineering in E. coli > (admittedly, coli transformation is artificially enhanced > in the laboratory), Naturally occurring transformation is limited to a small subset of bacteria. Streptococcus Pneumonia (formerly Pneumococcus, or also Diplococcus Pneumoniae) is one of them. It also occurs in Haemophilus influenza, but a certain sequence is required, so it preferentially picks up its own DNA (or that of a related strain). Bacillus subtilis also undergoes natural transformation. No more than a handful of other organisms do. Escherichia coli, the darling of molecular biologists does not undergo natural transformation. It was known for many years that DNA could be introduced by spheroplasting cells, but regeneration of bacteria was inefficient. It was only in 1973 that a method (the CaCl2 at 0C technique) was demonstrated that allows high efficiency DNA transfer into E. coli. This is far from natural, and less than half of the cells remain viable after treatment. Still with 10^10 cells/ml of transforming mixture, factors of 2 aren't really significant. Douglas Hanahan, more recently, has described methods using Ca, Mn, Cobalt Hexamine Chloride, DMSO, and DTT, and other reagants that I can't recall right off, that boost transformation frequencies several orders of magnitude - to almost a percent or higher. -- Craig Werner (MD/PhD '91) !philabs!aecom!werner (1935-14E Eastchester Rd., Bronx NY 10461, 212-931-2517) Everything's different. Nothing's changed. Well, only maybe slightly rearranged.
eddy@boulder.UUCP (06/02/87)
In article <1105@aecom.YU.EDU> werner@aecom.YU.EDU (Craig Werner) writes: > Naturally occurring transformation is limited to a small subset >of bacteria. Streptococcus Pneumonia (formerly Pneumococcus, or also >Diplococcus Pneumoniae) is one of them. > It also occurs in Haemophilus influenza, but a certain sequence >is required, so it preferentially picks up its own DNA (or that of >a related strain). > Bacillus subtilis also undergoes natural transformation. > > No more than a handful of other organisms do. > > Escherichia coli, the darling of molecular biologists does >not undergo natural transformation. It was known for many years that >DNA could be introduced by spheroplasting cells, but regeneration >of bacteria was inefficient. Sorry for being a disbelieving soul, but I beg to differ. According to my information, the following is a partial list of bacteria shown to undergo natural transformation (these are genera, not species): Bacillus, Escherichia, Hemophilus, Micrococcus, Mycoplasma, Neisseria, Proteus, Pseudomonas, Rhizobium, Staphylococcus, Streptococcus, Streptomyces, and Xanthomonas. Want to get more interesting? Consider eukaryotic cells that can be transformed...like human and mouse (it's called transfection when it's on cultured animal cells - while these days transfection efficiency is artificially increased by treatment with DEAE- dextran or some such reagent, transfection can occur without any particularly vicious cell treatment). I hardly consider this 'a handful of organisms.' And E. coli certainly *has* been reported to undergo natural transformation. Generally, molecular genetics techniques are not new tricks; they merely increase the efficiency of something nature's already been playing with. Perhaps our differences here, Craig, are ones of semantics and not science. (i.e. are bacteria on petri plates in the lab really undergoing 'natural' transformation?) My list, unless I seriously misunderstood the text, refers to bacteria in which the mere mixing of bacterial cultures with exogenous DNA fragments (usually from the same species) results in uptake & integration of the exogenous DNA at some detectable frequency. - Sean Eddy - Dept. of Molecular, Cellular, Developmental Biology - Univ. of Colorado, Boulder; Boulder, CO 80309 - eddy@Boulder.Colorado.EDU !{hao,nbires}!boulder!eddy - - "It all seemed to make some sort of sense at the time." - - Arthur Dent
gallagher@husc4.UUCP (06/03/87)
One question that interests me is this (I don't know the answer): If you define a species as a group of populations among which genetic information is freely exchanged, then what are the species of bacteria? Maybe the whole kingdom is a single species by this definition?! Paul Gallagher
werner@aecom.UUCP (06/03/87)
> Perhaps our differences here, Craig, are ones of semantics > and not science. (i.e. are bacteria on petri plates in the > lab really undergoing 'natural' transformation?) > - Sean Eddy Let's just say they are semantic differences and drop it. Suffice it to say that foreign DNA can be introduced into almost any organism, either naturally, or through a variety of artificial means, some mild (like Calcium Chloride), some harsh (like the recently described shot-gun technique for plant cells. There are a variety of species that foreign DNA cannot be introduced into, but if somebody wants to do it bad enough, they'll devise the methodology eventually. -- Craig Werner (MD/PhD '91) !philabs!aecom!werner (1935-14E Eastchester Rd., Bronx NY 10461, 212-931-2517) "Beware of Yuppies bearing Uzis."
eddy@boulder.UUCP (06/03/87)
In article <2172@husc6.UUCP> gallagher@husc4.UUCP (paul gallagher) writes: >One question that interests me is this (I don't know the answer): >If you define a species as a group of populations among which genetic >information is freely exchanged, then what are the species of bacteria? >Maybe the whole kingdom is a single species by this definition?! Answer: you *don't* define a species as a group of populations among which genetic information is freely exchanged. Too many examples exist in the animal world (and horrendous numbers in the plant world) of so-called 'species' inter-breeding; don't worry about the bacteria! Question: How *does* one define species? On the "seemed like a good idea at the time" principle? I really don't believe there's a good, hard rule; I'd like to hear one. (Technically, I don't believe all the bacteria exchange information. Most cases of 'natural' bacteria transformation involve a live bacterium of one species picking up exogenous DNA from a bacterium of the same species, and incorporating that DNA by homologous recombination. Some species, like Craig's example of Hemophilus influenzae, are so picky they will only a small fraction of the DNA from their own species. In any case, I believe the restrictions imposed by homologous recombination would tend to prevent free genetic exchange between all but the most closely related bacteria.) - Sean Eddy - Dept. of Molecular, Cellular, Developmental Biology - Univ. of Colorado, Boulder; Boulder, CO 80309 - eddy@Boulder.Colorado.EDU !{hao,nbires}!boulder!eddy - - "It all seemed to make some sort of sense at the time." - - Arthur Dent
srp@ethz.UUCP (06/04/87)
In article <1105@aecom.YU.EDU> werner@aecom.YU.EDU (Craig Werner) writes: > Escherichia coli, the darling of molecular biologists does >not undergo natural transformation. It was known for many years that >DNA could be introduced by spheroplasting cells, but regeneration >of bacteria was inefficient. > It was only in 1973 that a method (the CaCl2 at 0C technique) >was demonstrated that allows high efficiency DNA transfer into E. coli. >This is far from natural, and less than half of the cells remain >viable after treatment. Still with 10^10 cells/ml of transforming >mixture, factors of 2 aren't really significant. Douglas Hanahan, >more recently, has described methods using Ca, Mn, Cobalt Hexamine >Chloride, DMSO, and DTT, and other reagants that I can't recall right >off, that boost transformation frequencies several orders of magnitude - >to almost a percent or higher. You got most of the reagents, however even though there is a table reporting accross the board success with 'normal' strains in the paper, in my hands (and hands of other lab members), the procedure works "several orders of magnitude" better only with his own strains, the DH series (which, BTW, are _rumored_ to spontaneously mutate/revert or be otherwise unstable, even though it is RecA-!!). The technique does have the advantage of being a one-day procedure, whereas there is a 24 Hr. wait for the CaCl2 procedure (for competency to set in). It should be pointed out also that transformation efficency is also related to size of DNA being transformed, in a logarithmic relationship. Regards, Scott Presnell (Scientific Mercenaries Inc.) Organic Chemistry Swiss Federal Institute of Technology (ETH-Zentrum) CH-8092 Zurich, Switzerland. uucp:seismo!mcvax!cernvax!ethz!srp (srp@ethz.uucp); bitnet:Benner@CZHETH5A
pell@boulder.Colorado.EDU (Anthony Pelletier) (06/05/87)
(Scott Presnell) writes: re: hanahan protocol > >You got most of the reagents, however even though there is a table >reporting accross the board success with 'normal' strains in the paper, in >my hands (and hands of other lab members), the procedure works "several >orders of magnitude" better only with his own strains, the DH series >(which, BTW, are _rumored_ to spontaneously mutate/revert or be otherwise >unstable, even though it is RecA-!!). > Even Doug admits that the protocol works that much better only on the DH cell lines. For reasons that have never been clear to me, he does get the protocol to work on many other cell lines better than I (or anyone I know) have been able. Something in his body temperature or "karma" I guess. It is well established that HB101 will not stand up to the Hanahan proceedure. It even says so in the "Joy of Cloning." Concerning the _rumor_: I use DH1s mostly. I have had little trouble with that. But then I really don't care about the cell line so long as it transforms well and doesn't screw up my clones. I do routinely re-purify the strain before preparing a large batch of competent cells. >The technique does have the advantage of being a one-day procedure, >whereas there is a 24 Hr. wait for the CaCl2 procedure (for competency to >set in). > Two points: In HB101 and others I have used with the CaCl2 proceedure, the cells are competent immedeatly after treatment. I usually get 5-10 fold increase in competence if I let them sit for a day. I suppose it depends upon how many colonies you need as to whether the extra log is worth it. Second, competent cells can be stored frozen for months. I'd be glad to send a protocol if you like; but it is fairly intuitive. Just freeze in pre-cooled tubes in aliquots of .5-1.0 mls right in the CaCl2. Thaw on ice and use soon after thawing. We keep competent stocks of several cell lines and have had no trouble with any. It is possible that your work requires greater efficiency than mine, but if not, these changes will save you a fair bit of time. happy cloning. A.J.P. (Few degrees are worth remembering--and none are worth predicting) is is is
chiaraviglio@husc4.HARVARD.EDU (lucius) (06/06/87)
In article <105@bernina.UUCP> srp@bernina.UUCP (Scott Presnell) writes: >The technique I presume you mean the Hexamine Cobalt Chloride procedure? > does have the advantage of being a one-day procedure, >whereas there is a 24 Hr. wait for the CaCl2 procedure (for competency to >set in). I have run K38 E. Coli (which are HfrC Lamda-Lysogen, but otherwise wild-type, and give trashy transformation efficiency in general) through the cold CaCl[2] procedure for inducing competence and transformed them almost immediately after doing this and they are competent. You have to plate a load of them to get a reasonable number of colonies, but waiting a day or more neither helps nor hurts. (I was doing this because I had been told that even though freezing didn't kill them it robbed them of their competency over a short time in frozen storage, but this seems to be wrong or at least exaggerated.) Supposedly the freezing process increases the competency of some strains, but I have never tested this. Anyway, if you're in a hurry and using the CaCl[2] procedure, don't bother to wait 24 hours. If you have a strain that you know is helped by the freezing, just dump them in the liquid nitrogen and then thaw them out and use them right then. -- Lucius Chiaraviglio lucius%tardis@harvard.harvard.edu seismo!tardis.harvard.edu!lucius
werner@aecom.YU.EDU (Craig Werner) (06/07/87)
In article <1302@sigi.Colorado.EDU>, pell@boulder.Colorado.EDU (Anthony Pelletier) writes: > Even Doug admits that the protocol works that much better only on the > DH cell lines. For reasons that have never been clear to me, he does get > the protocol to work on many other cell lines better than I (or anyone I know) > have been able. Something in his body temperature or "karma" I guess. > It is well established that HB101 will not stand up to the Hanahan proceedure. > It even says so in the "Joy of Cloning." I have gotten Hanahan transformation to work with HB101, as well as JM101. JM83 failed miserably. I now use DH5alpha, as it has the lacZ complementation of the JM series, but comes hyped. On my best day, I got about 20% of Hanahan's maximum yield and that was fresh. Normally, I don't care, and use it frozen, and I get 5-10% of theoretical. (Frozen also omits DTT) Nowadays I clone into phage. It's much better for my purposes, and it is much more efficient by anybody's standards, which is 10-100 fold. -- Craig Werner (MD/PhD '91) !philabs!aecom!werner (1935-14E Eastchester Rd., Bronx NY 10461, 212-931-2517) "If I don't see you soon, I'll see you later."
bchso@uhnix2.UUCP (06/09/87)
In article <1211@sigi.Colorado.EDU> eddy@boulder.Colorado.EDU (Sean Eddy) writes: >In article <382@uhnix2.UUCP> bchso@uhnix2.UUCP (Dan Davison) writes: >>And via viruses. Bacillus sp. also pick up DNA directly from the environment, >>ie from those around them that have died and lysed the cell membrane. >Dan, is it true that only the Bacillus species can pick up >and incorporate DNA from the environment? >- Sean Eddy The classic transformation experiments were all done in Gram positive bacteria. It was first discovered in 1928 by F. Griffin, in Streptococcus pneumoniae. To quote Stent, from 'Molecular Genetics, 2nd Ed.': "For twenty-six years following Avery's identification of DNA as the transforming principle all efforts failed to demonstrate transformation in E. coli, the very bacterium with which the most extensive genetic and biochemical studies were being carried out during that time. Finally, in 1970, these efforts succeeded..." (pg. 197-8). For more than you'd ever want to know about the subject, see Lewin's Gene Expression 1 or Notani and Setlow, "Mechanism of bacterial transformation and transfection", Progress in Nucleic Acid Research and Molecular Biology Vol. 14:39 1974. If you peruse the section in Joy of Cloning (usually called Molecular Cloning by Maniatis et al.) they present three methods for transforming E. coli. The three could be descibed as tactical nuclear, intermediate range nuclear, and global nuclear war on E. coli membranes. I'm continually amazed that *any* bacteria survive such treatment. I know of no recent studies demonstrating Gram-negative transformation in natural populations. I suspect the problem is all the extra goodies contained in the outer membrane of Gram-negatives, not to mention the area between the inner and outer membranes (the periplasmic space). dr. dan davison/ Dept of Biochemical and Biophysical Sciences/ U. of Houston bitnet: bchs6\@uhupvm1.bitnet | 4800 Calhoun/ Houston, Tx 77004 arpanet: davison\@sumex-aim.stanford.edu|uucp:...rice!academ!uhnix1!uhnix2!bchso after July 1: T-10 MS K-710, Los Alamos National Laboratory, Los Alamos, N.M. 87545 (Theoretical Biology Division)
bchso@uhnix2.UUCP (06/09/87)
In article <1105@aecom.YU.EDU> werner@aecom.YU.EDU (Craig Werner) writes: > Douglas Hanahan, >more recently, has described methods using Ca, Mn, Cobalt Hexamine >Chloride, DMSO, and DTT, and other reagants that I can't recall right >off, that boost transformation frequencies several orders of magnitude - >to almost a percent or higher. I was a grad student at SUNY Stony Brook and had occasion to hang out at the Cold Spring Harbor Laboratory from time to time. Doug Hanahan worked there and before he published the recipe the reagent mix mentioned by proto-doctor Werner was commonly referred to as "liquid gold". dr. dan davison/ Dept of Biochemical and Biophysical Sciences/ U. of Houston bitnet: bchs6\@uhupvm1.bitnet | 4800 Calhoun/ Houston, Tx 77004 arpanet: davison\@sumex-aim.stanford.edu|uucp:...rice!academ!uhnix1!uhnix2!bchso After July 1: T-10 MS K-710 Los Alamos National Laboratory, Los Alamos N.M. 87545 (Division of Theoretical Biology)
bchso@uhnix2.UUCP (06/09/87)
In article <1247@sigi.Colorado.EDU> eddy@boulder.Colorado.EDU (Sean Eddy) writes: >> No more than a handful of other organisms do. >> >Sorry for being a disbelieving soul, but I beg to differ. >According to my information, the following is a partial list >of bacteria shown to undergo natural transformation (these are >genera, not species): [list of genera edited out] This list reads like the one from Levinthal, Ann. Rev. Microbiology 28:219- 230 1974. Is it? Or is it updated? >I hardly consider this 'a handful of organisms.' Gee, Sean, I consider this a handful. But this is a difference in philosophy, since the mammalian cell culture transformation you cite would never be the same transformation as bacterial, *to me*. >And E. coli certainly *has* been reported to undergo natural >transformation. References please? >unless I seriously misunderstood the text, [it] refers to bacteria >in which the mere mixing of bacterial cultures with exogenous >DNA fragments (usually from the same species) results in >uptake & integration of the exogenous DNA at some detectable >frequency. This sounds a little like 'mystery transformation' from the Rhodopseudomonas. Doug Youvan (see this month's Scientific American, the mechanism of photo- synthesis) has used a technique in which you mix bacteria on a plate then put them in an incubator overnight (with candlelight and music) and magically a few days later the genes have been transferred to the recipient. As near as can be figured out, last I knew, the speculation was that one of the three bacteria you had to add to the mix (donor, recipient, and helper) actually mediated the transfer. Mixing donor and recipient together provided a nice negative control. I think this was suspected to be conjugative...does anyone know differently? dr. dan davison/ Dept of Biochemical and Biophysical Sciences/ U. of Houston bitnet: bchs6\@uhupvm1.bitnet | 4800 Calhoun/ Houston, Tx 77004 arpanet: davison\@sumex-aim.stanford.edu|uucp:...rice!academ!uhnix1!uhnix2!bchso After July 1: T-10 MS K-710 Los Alamos National Lab, Los Alamos N.M. 87545, Division of Theoretical Biology
pell@boulder.Colorado.EDU (Anthony Pelletier) (06/09/87)
i wrote: It is well established that HB101 will not stand up to the Hanahan proceedure. It even says so in the "Joy of Cloning." > craig wrote: > I have gotten Hanahan transformation to work with HB101, as well >as JM101. > This will teach me not to believe what I read. Even the cloning manual can be wrong. It said not to try it on HB101 and I didn't. The rest of what I said was from first-hand experience and therefore should more closely approximate the truth. Thanks for the info, and mea culpa. Also, thanks for the info regarding ph. I never checked it before. tony (Empire Truck Driving School grad--cum laude '92)
eddy@boulder.Colorado.EDU (Sean Eddy) (06/11/87)
In article <394@uhnix2.UUCP> bchso@uhnix2.UUCP (Dan Davison) writes: >In <1247@sigi.Colorado.EDU> eddy@boulder.Colorado.EDU (Sean Eddy) writes: >This list reads like the one from Levinthal, Ann. Rev. Microbiology 28:219- >230 1974. Is it? Or is it updated? No, the first thing I laid my hands on was a CRC Handbook of Microbiology. >Gee, Sean, I consider this a handful. But this is a difference in >philosophy, since the mammalian cell culture transformation you cite >would never be the same transformation as bacterial, *to me*. Why not? I don't mean 'growth transformation' or 'neoplastic transformation', I'm just referring to the uptake and integration of exogenous DNA fragments. >>And E. coli certainly *has* been reported to undergo natural >>transformation. > >References please? Uh-oh, can't find my notes... what I saw was an old article in Nature, and was referenced in the CRC along with all the other genera that are reported to undergo natural transformation. > >>unless I seriously misunderstood the text, [it] refers to bacteria >>in which the mere mixing of bacterial cultures with exogenous >>DNA fragments (usually from the same species) results in >>uptake & integration of the exogenous DNA at some detectable >>frequency. > >This sounds a little like 'mystery transformation' from the Rhodopseudomonas. >Doug Youvan (see this month's Scientific American, the mechanism of photo- >synthesis) has used a technique in which you mix bacteria on a plate then >put them in an incubator overnight (with candlelight and music) and magically >a few days later the genes have been transferred to the recipient. As near as >can be figured out, last I knew, the speculation was that one of the three >bacteria you had to add to the mix (donor, recipient, and helper) actually >mediated the transfer. Mixing donor and recipient together provided a nice >negative control. I think this was suspected to be conjugative...does anyone >know differently? > No, I was just trying (clumsily, I guess) to define bacterial transformation. In the lab, you pretreat the bacteria (make competent cells with CaCl2) and then mix them with the DNA you want them to take up. I was trying to say that you might call 'natural transformation' cases in which the pretreatment is not absolutely necessary. - Sean Eddy - MCD Biology; U. of Colorado at Boulder; Boulder CO 80309 - eddy@boulder.colorado.EDU !{hao,nbires}!boulder!eddy - - "Why should the government subsidize intellectual curiosity?" - - Ronald Reagan
werner@aecom.UUCP (06/13/87)
In article <393@uhnix2.UUCP>, bchso@uhnix2.UUCP (Dan Davison) writes: > > I was a grad student at SUNY Stony Brook and had occasion to hang out at > the Cold Spring Harbor Laboratory from time to time. Doug Hanahan > worked there and before he published the recipe the reagent mix mentioned > by proto-doctor Werner was commonly referred to as "liquid gold". > Actually, there's more to it than that. The combination of MnCl2 (which is slightly pinkish) and the Hexamine Cobalt Chloride actually lead to a transformation buffer which can only be described as a brilliant gold color. -- Craig Werner (MD/PhD '91) !philabs!aecom!werner (1935-14E Eastchester Rd., Bronx NY 10461, 212-931-2517) "Illness strips away superficiality to reveal reality in etched detail."