A short history of the axenic strains of Dictyostelium discoideum

Sussman and Sussman (1967; d3234) report the first isolation of an axenically growing strain Ax-1 by sub-culturing of their lab strain DdB (NC-4) in an HL5-like medium containing liver extract and fetal calf serum. There is no mention of mutagens. DdB is an NC-4 derivative selected by the continuous sub-culturing of colonies that showed more synchronous development and less spreading colony morphology. The first mentioning of DdB is by Brackenbury et al. (1974; d0154).

Both Schwalb and Roth (1970; d1394) and Watts and Ashworth (1970; d2187) eliminate the serum and liver extract from the medium by prolonged sub-culturing of Ax-1. Again, there is no mention of mutagens. Watts and Ashworth call the isolate, which can grow in the simplified medium, Ax-2 (AX2). Ax-1 seems to have been lost.

Cocucci and Sussman (1970; d0244) use the term HL-5 (HL5) medium to describe the simplified axenic medium.

Loomis (1971; d2992) isolates independently an axenic strain with the use of N-methyl-Ni-nitro-N-nitrosoguanidine, and calls this strain A3 (AX3).

Williams et al. (1974; d1988) establish that at least two recessive mutations (axeA on linkage group II and axeB on linkage group III) are involved in axenic growth. North and Williams (1978; d1164) establish that a third locus axeC on linkage group II is involved in the axenic growth of strains AX2 and AX3.

Franke and Kessin (1977; d0473) develop a defined minimal medium (FM) for the axenic strain AX3 (AX2 grows well in it too). Their axenic strain probably came by way of Frank Rothmanis laboratory. The same article contains a simplified recipe for HL5. Be careful though: the modified HL5 recipe is less concentrated than the original version, but shows the same growth kinetics. Occasionally a version of HL5 containing maltose rather than glucose is used (Cosson et al. 2002; d7294 and Korohada et al. 2002; d7353). A more detailed protocol on how to prepare FM can be found here.

The Kessin lab AX3 strain has since been renamed AX3-K or AX4 (Knecht et al. 1986; d0876).

Jakob Franke, 7/30/2003

Dictyostelium Strain History discussion

Date: Tue, 2 Nov 1999 14:41:46 -0600

Reply-To: Dicty@listserv.ACNS.NWU.EDU

Sender: owner-Dicty@listserv.ACNS.NWU.EDU

X-Phforward: V2.0@iris

From: richard sucgang phd <rsucgang@bcm.tmc.edu>

To: Dicty@listserv.ACNS.NWU.EDU

 

Chris LaRosa contacted me with some questions about the genealogy of the commonly used axenic strains, and I answered to the best of my knowledge, but I would like to throw the question and answers out to the people who may have a better recall of the history:

 

NC-4 --> Ax1 (Sussman and Sussman)

Ax1 --> Ax2 (Watts and Ashworth)

NC-4 --> Ax3 (Loomis)

Ax3 --> Ax3K / aka KAx3 (Franke and Kessin)

Ax3K --> Ax4 (???)

 

Does this look right? It would be nice to hear from whoever can lay claim to first naming Ax4.

 

-ricky

 

 

Date: Tue, 2 Nov 1999 13:21:26 -0800

Reply-To: Dicty@listserv.ACNS.NWU.EDU

Sender: owner-Dicty@listserv.ACNS.NWU.EDU

X-Phforward: V2.0@iris

From: Rick Firtel <rafirtel@ucsd.edu>

To: Dicty@listserv.ACNS.NWU.EDU

Subject: Re: Strain History

 

I think think that KAx-3 (Ax-3K) and Ax-4 are the same. I think Bill didn't like the Ax-3 name.

 

Best-

 

Rick

 

 

Date: Tue, 02 Nov 1999 16:48:20 -0500

Reply-To: Dicty@listserv.ACNS.NWU.EDU

Sender: owner-Dicty@listserv.ACNS.NWU.EDU

X-Phforward: V2.0@iris

From: Richard Kessin <rhk2@columbia.edu>

To: Dicty@listserv.ACNS.NWU.EDU

Subject: Re: Strain History

 

Dear Colleagues,

According to our records, which are pretty good, we sent strain AX3(1), which has been stored in our lab since 1974 to Bill in June of 1980. Bill, thinking it looked different from other Ax3s, named in AX4. We sent the strain to Rick at the same time and he named it KAX3, for Kessin Ax3. The strain has been maintained in silica gel by Jakob Franke for the last 25 years and not by Rich Kessin, who nevertheless got the credit, thanks to Rick. I am glad to set the record straight. Neither Jakob nor I can remember where we got our original AX3 strain.

 

Rich Kessin

 

 

 

Date: Tue, 2 Nov 1999 14:17:50 -0800

Reply-To: Dicty@listserv.ACNS.NWU.EDU

Sender: owner-Dicty@listserv.ACNS.NWU.EDU

X-Phforward: V2.0@iris

From: wloomis@ucsd.edu

To: Dicty@listserv.ACNS.NWU.EDU

Subject: Re: Strain History

 

In 1984 or 1985 Dave Knecht used a stock of AX3 that we received back from Rich Kessin and selected for cells that transfomed well. He then picked a small, tight plaque and named it AX4 to keep it straight from other strains. This is the strain that we mapped and is now being sequenced.

 

While I was writing this I read Rich Kessin's e-mail on this subject. I am fairly sure that I sent him AX3 while he was at Harvard. He then took it to Columbia and it wended its way back here in 1980. It was one of the stocks that we tested in 1985 and seemed a good strain for transformation.

 

In my opinion the name AX4 is cleaner than KAx-3, KAX 3, AK3k etc. There are many lyophilized stocks kept here.

 

Bill

 

Date: Wed, 03 Nov 1999 08:29:57

Reply-To: Dicty@listserv.ACNS.NWU.EDU

Sender: owner-Dicty@listserv.ACNS.NWU.EDU

X-Phforward: V2.0@iris

From: "Prof. Wolfgang Nellen" <nellen@hrz.uni-kassel.de>

To: Dicty@listserv.ACNS.NWU.EDU

Subject: Re: Strain History

 

just for the record:

 

I believe the first genetic difference between "AX3-K" (AX4) and "AX3-L" (AX3) was found by Steve Poole in Rick's lab when he looked at the discoidin genes and sourrounding Tdds (repetitive elements).

 

regards

wolfgang

 

Date: Wed, 3 Nov 1999 11:47:04 +0000

Reply-To: Dicty@listserv.ACNS.NWU.EDU

Sender: owner-Dicty@listserv.ACNS.NWU.EDU

X-Phforward: V2.0@iris

From: Jeff Williams <j.g.williams@dundee.ac.uk>

To: Dicty@listserv.ACNS.NWU.EDU

Subject: Re: Strain History

 

I donit want to get involved in the origins of the Ax4 strain discussion but I have to respond to Bill Loomis's statement that the genome of Ax4 is now being sequenced. The decision to chose Ax4 over Ax2 was hotly debated among those of us involved in the genome project and the deciding factor was the availability of the YAC map for Ax4. Ax2 has significant experimental and biological advantages over Ax3(4) and Ax4 has a large duplication in its genome that will cost us extra effort. I am not trying to re-open a closed issue but I think it important that new comers to field understand that Ax2 holds many advantages - we should not be locked into Ax4 just because we will soon know its sequence. At the level of the individual gene differences with Ax4 will be very rare, only chromosomal organisation is likely to differ significantly.

 

Jeff Willaim

 

 

Date: Wed, 3 Nov 1999 08:51:11 -0600

Reply-To: Dicty@listserv.ACNS.NWU.EDU

Sender: owner-Dicty@listserv.ACNS.NWU.EDU

X-Phforward: V2.0@iris

From: "Rex L. Chisholm" <r-chisholm@nwu.edu>

To: <Dicty@listserv.ACNS.NWU.EDU>

Subject: RE: Strain History

 

Hi Jeff,

 

I would find it interesting if you could list some of the experimental and biological advantages that Ax2 offers. Perhaps it would help us understand some of the differences.

 

Rex

 

 

Date: Wed, 3 Nov 1999 17:01:17 +0000

Reply-To: Dicty@listserv.ACNS.NWU.EDU

Sender: owner-Dicty@listserv.ACNS.NWU.EDU

X-Phforward: V2.0@iris

From: Jeff Williams <j.g.williams@dundee.ac.uk>

To: Dicty@listserv.ACNS.NWU.EDU

Subject: RE: Strain History

 

 

Dear Rex

 

I know why we prefer Ax2 but rather than give a limited list perhaps we should poll the other groups and try to come up with a comprensive comparison with Ax3. It could be a very useful exercise.

 

Jeff

 

Date: Wed, 03 Nov 1999 17:51:43 +0100

Reply-To: Dicty@listserv.ACNS.NWU.EDU

Sender: owner-Dicty@listserv.ACNS.NWU.EDU

X-Phforward: V2.0@iris

From: Pierre Cosson <Pierre.Cosson@medecine.unige.ch>

To: Dicty@listserv.ACNS.NWU.EDU

Subject: RE: Strain History

 

 

Dear Everybody,

 

Since we are on this subject, does anyone know the relationship of the DH1 strain with the other strains ?

 

Thanks

 

Pierre Cosson

 

Date: Wed, 3 Nov 1999 09:01:16 -0800

Reply-To: Dicty@listserv.ACNS.NWU.EDU

Sender: owner-Dicty@listserv.ACNS.NWU.EDU

X-Phforward: V2.0@iris

From: wloomis@ucsd.edu

To: Dicty@listserv.ACNS.NWU.EDU

Subject: Re: Strain History

 

Jeff,

 

I don't think it helps to focus on the 300 kb duplication on chromosome 2 that we found in AX4 but not in AX2. How many duplications are present in AX2 and absent in AX4?

 

A more important point is that the microbial life style of Dicty leads to the accumulation of random chromosomal changes during serial passage. People like David Soll are well aware that it is essential to use a standard stock and not sub-culture more than necessary. The AX2 strain that you use and like may not be genetically the same as another stock called AX2 in another lab. We should keep genetic uniformity in mind as we push our analyses to more subtle processes.

 

Bill

 

Date: Wed, 3 Nov 1999 11:22:11 -0600

Reply-To: Dicty@listserv.ACNS.NWU.EDU

Sender: owner-Dicty@listserv.ACNS.NWU.EDU

X-Phforward: V2.0@iris

From: "Rex L. Chisholm" <r-chisholm@nwu.edu>

To: <Dicty@listserv.ACNS.NWU.EDU>

Subject: RE: Strain History

 

Jeff,

 

I agree it could be a useful exercise. I use Ax3 because that's what Harvey used. Not the best reason. Now we have a ton of strains with knockouts and various transgenes in Ax3 which is a better reason, but still not the best. Since you know why you like Ax2, why don't you start with your list. I'm sure everyone else will be stimulated to add their input. I'll collate everything and if it would be useful can even set up a web page that lists the advantages of each strain.

 

Rex

 

 

Date: Wed, 3 Nov 1999 18:30:57 +0100 (MET)

Reply-To: Dicty@listserv.ACNS.NWU.EDU

Sender: owner-Dicty@listserv.ACNS.NWU.EDU

X-Phforward: V2.0@iris

From: macw@zi.biologie.uni-muenchen.de (Harry MacWilliams)

To: Dicty@listserv.ACNS.NWU.EDU

Subject: Re: Strain History

 

Hi everyone,

 

If we are really looking for a standard, shouldnt we think about NC4? Aren't all axenic strains the results of long and little-understood selection procedures? And arent they all a little wierd, being as they go around phagocytosing liquid, and express development genes during growth?

 

Harry MacWilliams

 

Date: Wed, 3 Nov 1999 09:57:36 -0800

Reply-To: Dicty@listserv.ACNS.NWU.EDU

Sender: owner-Dicty@listserv.ACNS.NWU.EDU

X-Phforward: V2.0@iris

From: wloomis@ucsd.edu

To: Dicty@listserv.ACNS.NWU.EDU

Subject: Re: Strain History

 

Harry,

 

Of course you are right that the bench mark strain is Raper's NC4. Many years ago he gave me one of the first lyophils that he made in 1935. I keep it in a reliquary.

Keep in mind that axenic strains do not express developmental genes when they are grown the same way as NC4 ie. on bacterial lawns.

 

Bill

 

Date: Wed, 3 Nov 1999 12:04:47 -0600

Reply-To: Dicty@listserv.ACNS.NWU.EDU

Sender: owner-Dicty@listserv.ACNS.NWU.EDU

X-Phforward: V2.0@iris

From: "Rex L. Chisholm" <r-chisholm@nwu.edu>

To: <Dicty@listserv.ACNS.NWU.EDU>

Subject: RE: Strain History

 

I'm not sure we should be looking for a standard. We're probably too far down too many different roads for that. To my thinking the more valuable would be to understand the differences and any resulting strengths and weaknesses. Regarding NC4, what is the status of molecular genetic manipulation of NC4? Any successful knockouts? How easily is NC4 transformed?

 

Date: Wed, 03 Nov 1999 19:07:53 +0100

Reply-To: Dicty@listserv.ACNS.NWU.EDU

Sender: owner-Dicty@listserv.ACNS.NWU.EDU

X-Phforward: V2.0@iris

From: Michael Schleicher <Michael.Schleicher@bio.med.uni-muenchen.de>

To: Dicty@listserv.ACNS.NWU.EDU

Subject: RE: Strain History

 

Hi Rex,

 

I like your sentence "I use AX3 because that's what Harvey used...". Perhaps in some sort of sentimental way I like AX2 because I trust the strain. During my time with Guenther Gerisch I learned how rigorous one has to be to keep biological material clean and homogeneous. Ages ago (sorry HERR GERISCH), Guenther Gerisch recloned AX2 (clone 214, therefore in his papers "AX2-214"). This was and still is the "holy stock" of spores in his freezer. Once a year one aliquot was taken from this untouchable stock and analyzed for growth in axenic medium and on bacteria, for the strain's ability to develop in exactly the same well known time frame, in its expression of contact site A and other markers during early and later hours of development etc. etc. Only if the material was flawless, a batch of about 100 aliquots of spores was prepared and stored in liquid nitrogen. Once a week during the following year fresh aliquots from this batch were used for experiments. After about one year the whole procedure was repeated. I try to be as rigorous with my AX2 as I learned it from Guenther Gerisch.

His care for a good AX2 strain is reason enough (for me) to believe in AX2 as the "wild type" in our genetic work. If I recall correctly, Richard Kessin treated his AX3 in a similar way as Guenther Gerisch did. I was a little bit shocked when I read Bill's statement about the different AX2 strains in Europe. I do not agree.

 

Michael

 

 

Date: Wed, 3 Nov 1999 18:33:02 +0000

Reply-To: Dicty@listserv.ACNS.NWU.EDU

Sender: owner-Dicty@listserv.ACNS.NWU.EDU

X-Phforward: V2.0@iris

From: Robert Insall <R.H.Insall@bham.ac.uk>

To: Dicty@listserv.ACNS.NWU.EDU

Subject: RE: DH1

 

DH1 was made by Dale Hereld in the Devreotes lab. It's an AX3 parent with the entire coding sequence of pyr56 removed using a PCR-constructed splint and FOA selection.

 

Robert Insall

 

 

Date: Wed, 3 Nov 1999 11:48:34 -0800

Reply-To: Dicty@listserv.ACNS.NWU.EDU

Sender: owner-Dicty@listserv.ACNS.NWU.EDU

X-Phforward: V2.0@iris

From: Rick Firtel <rafirtel@ucsd.edu>

To: Dicty@listserv.ACNS.NWU.EDU

Subject: RE: Strain History

 

Dear Boys and Girls and children of all ages:

 

I think this has gotten a bit silly. There are reasons, historical or otherwise, for using different strains. Having collaborated with Jeff for a while, I see advantages for Ax2 on some things but like KAx-3 (our Ax-3 or Ax-4) for others (signaling during aggregation,

they self oscillate nicely). These type of chauvinistic comments helps no one and gets us no where. To try to suggest that everyone should shift to one strain or another is silly and not very congenial. I have thousands of strains in the lab as do other people and am not about to change and, as others have commented, many people are in the same situation. Maybe we can stop this now before others get pissed off.

 

Best-

 

Rick

 

 

 

Date: Wed, 3 Nov 1999 14:01:14 -0600

Reply-To: Dicty@listserv.ACNS.NWU.EDU

Sender: owner-Dicty@listserv.ACNS.NWU.EDU

X-Phforward: V2.0@iris

From: "Rex L. Chisholm" <r-chisholm@nwu.edu>

To: <Dicty@listserv.ACNS.NWU.EDU>

Subject: RE: Strain History

 

Rick,

 

I believe the discussion is potentially useful, but agree with you that no one is going to decide to change strains when they have a big investment. What I hope to learn is the advantages that keep getting referred to, including those you allude to in your message. It would help me make intelligent decisions about which strain to use for a particular experiment. I agree with you that this should not become a "mine is better than yours"

discussion. But I still would like to better understand the strengths and weaknesses of the main strains that people use--and this discussion could help accomplish that.

 

Best,

 

Rex

 

Date: Wed, 3 Nov 1999 15:08:21 -0500 (EST)

Reply-To: Dicty@listserv.ACNS.NWU.EDU

Sender: owner-Dicty@listserv.ACNS.NWU.EDU

X-Phforward: V2.0@iris

From: mxr37@madeline.INS.CWRU.Edu (Maribel Rico)

To: Dicty@listserv.ACNS.NWU.EDU

Subject: RE: Strain History

 

Yeah Rick,

 

This might be childish but now I got very curious about the dark origins of the several Dicty strains....and why one would use this rather than that. I agree it will be useful information and seem kind of inaccessible for me nowadays.

 

Maribel Rico

 

 

 

Date: Wed, 03 Nov 1999 15:29:39 -0500

Reply-To: Dicty@listserv.ACNS.NWU.EDU

Sender: owner-Dicty@listserv.ACNS.NWU.EDU

X-Phforward: V2.0@iris

From: Tom Egelhoff <tte@po.cwru.edu>

To: Dicty@listserv.ACNS.NWU.EDU

Subject: RE: Strain History

 

David Knecht should weigh in on the NC4 issue. He isolated a spontaneous axenic isolate of NC4 (bravely renamed NC4-A2) that we have been working with. It grows happily in HL5, fruits robustly (i.e. large fruiting bodies, like NC4 and unlike Ax2 & 3 which for us give much smaller fruiting bodies), and I believe it is transformable, but Knecht would need to speak to that point.

 

Tom Egelhoff

 

 

 

Date: Wed, 3 Nov 1999 19:31:59 -0500 (EST)

Reply-To: Dicty@listserv.ACNS.NWU.EDU

Sender: owner-Dicty@listserv.ACNS.NWU.EDU

X-Phforward: V2.0@iris

From: Jakob Franke <jf31@columbia.edu>

To: Dicty@listserv.ACNS.NWU.EDU

Subject: Re: Strain History

 

The stream of strain history messages has become so large, I may as well add to it.

 

In: Meth. Cell Biol. Vol. 28, page 13 (1987), Maurice Sussman writes a short history of the axenic strains. The parental strain was DdB, which was his lab strain. DdB also was the strain Bill Loomis used. DdB did not look much like the original NC-4 that I saw many years later.

 

The history Raper gives (pg. 73-75 of his book) is a bit more precise and indicates that AX2 was a derivative of AX1.

 

'Our' strain AX4 (AX3K) was a clone of AX3 that was used to develop the minimal medium (FM).

 

The genealogy would look like this:

 

NC-4 --> DdB --> AX1 --> AX2 --> AX3 --> AX4 = AX3K

 

Jakob Franke.

 

Date: Wed, 3 Nov 1999 16:52:35 -0800

Reply-To: Dicty@listserv.ACNS.NWU.EDU

Sender: owner-Dicty@listserv.ACNS.NWU.EDU

X-Phforward: V2.0@iris

From: Rick Firtel <rafirtel@ucsd.edu>

To: Dicty@listserv.ACNS.NWU.EDU

Subject: Re: Strain History

 

 

J-

 

Thanks for clarifying what seemed to be muddy waters and establishing the genealogy. I also think that KAx-3 (Ax-3K) and Ax-4 are essentially the same strain, especially as wild-type strains used to travel around the building and back and forth across the street in La Jolla, although they have been separate for the last 5-8 years. As long as people define their strains in papers, it should be clear to all.

 

Best-

 

Rick

 

 

Date: Thu, 04 Nov 1999 09:56:15

Reply-To: Dicty@listserv.ACNS.NWU.EDU

Sender: owner-Dicty@listserv.ACNS.NWU.EDU

X-Phforward: V2.0@iris

From: "Prof. Wolfgang Nellen" <nellen@hrz.uni-kassel.de>

To: Dicty@listserv.ACNS.NWU.EDU

Subject: RE: Strain History

 

I agree with Michael and assume that at least the "Gerisch-offspring" keeps to the ritual!

 

to start with differences:

 

we used AX4 and AX2 to look to cAMP-pulse induced ALF transcription. Pulse induction is observed in AX3 (actually K-AX3 = AX4) but not in AX2. This is due (at least for ALF) to higher endogenous cAMP production of AX2 so there is no response to additional pulses. (This is of course not true for csA, the Gerisch-group standard gene to check for cAMP responsiveness). Ref. is May et al., Mech. of Dev. 33, 147, 1991 and May et al., MCB 9, 4653, 1989.

 

regards

wolfgang

 

 

Date: Thu, 04 Nov 1999 10:05:21

Reply-To: Dicty@listserv.ACNS.NWU.EDU

Sender: owner-Dicty@listserv.ACNS.NWU.EDU

X-Phforward: V2.0@iris

From: "Prof. Wolfgang Nellen" <nellen@hrz.uni-kassel.de>

To: Dicty@listserv.ACNS.NWU.EDU

Subject: RE: Strain History

 

 

let's compile a page with strain differences anyway. I believe this is important to sort out seemingly contradicting data obtained with different strains! I agree with Rick that the use of different strains is very useful but to nail down these differences is also!

 

wolfgang

 

 

Date: Thu, 4 Nov 1999 10:09:41 +0100

Reply-To: Dicty@listserv.ACNS.NWU.EDU

Sender: owner-Dicty@listserv.ACNS.NWU.EDU

X-Phforward: V2.0@iris

From: wetterau@zi.biologie.uni-muenchen.de (Birgit Wetterauer)

To: Dicty@listserv.ACNS.NWU.EDU

Subject: RE: Strain History

 

NC4 can be transformed quite easily.

 

see

 

Wetterauer, B.W., Morandini, P., Hribar, I., Hamker, U., Singelton, R. and MacWilliams, H.K. (1996) Transformation of nonaxenic Dictyostelium discoideum strains using a new selection cassette. Plasmid 36, 169-181

 

Birgit Wetterauer

 

 

Date: Thu, 4 Nov 1999 12:12:51 +0000

Reply-To: Dicty@listserv.ACNS.NWU.EDU

Sender: owner-Dicty@listserv.ACNS.NWU.EDU

X-Phforward: V2.0@iris

From: Jeff Williams <j.g.williams@dundee.ac.uk>

To: Dicty@listserv.ACNS.NWU.EDU

Subject: Re: Strain History

 

Dear All,

 

If I can first explain why I wrote the original letter. I want to avoid a situation some years down the line when mine and other "European" papers are criticised for not using the "standard axenic strain" - there is no such thing; its just a question of "horses for courses". If you want a basic cell for cell biology or want to study early signalling Ax3 and its derivatives are fine. However, the Ax4 isolate we have used is clearly aberrant in some aspects of late development. The most dramatic example of this came from the collaboration Rick alluded to - to characterise the StatA REMI disruptant, that was isolated in Ax4. All the markers we have generated over the years were originally characterised in Ax2 but, working with Rick, we found significant differences between Ax2 and Ax4 and eventually had to re-generate the disruption in Ax2 and analyse the markers there. The most worrying difference was that the ecmB:lacZ reporter was expressed throughout the prestalk region of Ax4. In slugs of an Ax2 strain ecmB is a marker for commitment to stalk cell differentiation and is expressed in a cone of pstAB cells in the slug tip. We and John Sternfeld showed that the pstAB cells are the precursors of the inner basal disc. The important point is that John used wild type NC4 cells - so the Ax2 pattern is the correct one. To the non-cognoscenti this may all sound obtruse but it showsthat these Ax4 slugs have a prestalk zone composed of cells expressing a marker that is normally only expressed when a prestalk cell commits itself to stalk cell differentiation. This is presumably the tip of on an iceberg of mis-expression. I totally agree that Ax2 can be mis-handled, with bad consequences. I also agree that the parental strain NC4 is the benchmark and Bergit's EMAIL encourages me to try using it with the cell type markers. Adrian Harwood made a "minimal manipulation", axenic derivative of NC4, by simply selecting cells on plastic for growth in axenic medium, but we have never done side by side transformations with Ax2 to determine relative efficiencies. Adrian's strain would have been a good candidate for genome sequencing as it never suffered the mutagenesis that was used to generate Ax3 (Watts and Ashworth generated Ax2 by selecting unmutagenised cells). Ax3 has a duplication (Bill - In your 1995 Genetics paper it was 1Mb not 300Kb - did the subsequent YAC mapping reduce the estimate?) but Ax2 does not have that particular duplication. Once again, I am not trying to re-open a closed debate - Ax4 is being sequenced. The priority now is to make sure that is done as rapidly and accurately as joint international resources allow. What I think will be valuable is to summarise the Ax2/Ax4 differences so that we can all chose the best strain for our particular purposes.

 

Jeff

Date: Thu, 4 Nov 1999 10:00:51 -0500

Reply-To: Dicty@listserv.ACNS.NWU.EDU

Sender: owner-Dicty@listserv.ACNS.NWU.EDU

X-Phforward: V2.0@iris

From: "Mcnally, James (NCI)" <mcnallyj@dce41.nci.nih.gov>

To: "'Dicty@listserv.ACNS.NWU.EDU'" <Dicty@listserv.ACNS.NWU.EDU>

Subject: RE: Strain History

 

Regarding strain differences, we have compared morphogenesis and cell motion in

the AX-2 and KAX-3 strains used in our lab. We found the following differences:

 

AX-2 - aggregation was later, cell trajectories in the mound were a combination of random and radial, optical density waves were concentric target patterns, culmination tended to be direct (i.e. no migrating slug)

 

KAX-3 - aggregation was earlier, cell trajectories in the mound were random early and then rotational, optical density waves were rotating spirals, slugs tended to migrate before culminating.

 

The differences in culmination mode tendencies encouraged us to look at how altering buffering conditions could affect some of the other morphogenetic features we looked at. In particular we found that under the appropriate buffering conditions we could interchange not only slug migration mode, but also mound cell motion patterns (e.g. AX-2 mounds could exhibit rotational motion, and KAX-3 mounds could exhibit random and radial motion.)

 

Our conclusion was that development in general in Dictyostelium is plastic, and that this plasticity is retained in each of the strains, although each strain has different developmental tendencies. So we would also argue that with respect to morphogenesis, there is no "right strain" to work with, since each strain exhibits behavior that the other strain is capable of exhibiting. But for example, if one wants to specifically study rotational motion, then the KAX-3 strain is a better choice.

 

For details, see Kellerman and McNally, Develop. Biol. 208:416-429 (1999)

 

Jim

Date: Thu, 4 Nov 1999 09:42:01 -0800

Reply-To: Dicty@listserv.ACNS.NWU.EDU

Sender: owner-Dicty@listserv.ACNS.NWU.EDU

X-Phforward: V2.0@iris

From: wloomis@ucsd.edu

To: Dicty@listserv.ACNS.NWU.EDU

Subject: Re: Strain History

 

Jeff,

 

Concerning the possibility that ecmB is expressed in a different pattern in AX4 than in AX2, the in situ hybridization data of Ricardo Escalante shows the exact same pattern of ecmB in AX4 slugs that your lab had previously described in an AX2 slug. Namely, a central core of cells containing ecmB mRNA at the anterior of slugs. Take a look at Figures 2K and 2L in:

 

Escalante, R. and Loomis, W. F. (1995) Whole-mount in situ hybridization of

Cell-type Specific mRNAs in Dictyostelium. Devel. Biol. 171:262-266.

 

The strain that you and Rick were working with may have genetically diverged from AX4.

 

Our 1995 Genetics paper gave the size of the duplication from one end to the other - ie. 300 ± 200 kb on one side is tandemly duplication as an inversion on the other side. The ends of the duplication are not defined with an accuracy of greater than about 200 kb. Sequencing will provide the exact size and gene content of the 300 ± 200 kb duplication (about 0.1% of the genome).

 

Bill

 

Date: Fri, 5 Nov 1999 08:04:12 +0000

Reply-To: Dicty@listserv.ACNS.NWU.EDU

Sender: owner-Dicty@listserv.ACNS.NWU.EDU

X-Phforward: V2.0@iris

From: keith.williams@proteomesystems.com

To: Dicty@listserv.ACNS.NWU.EDU

Cc: keith.williams@proteomesystems.com

Subject: Re: Strain History

 

Dear Dicty freaks

 

Perhaps I can take a step further back for newcomers to the field and point out that there are other isolates!

 

I think it is interesting that the "type" strain, in our case NC4, does not always reflect the average out there in the wild, probably because the type strain almost always represents and early isolate that is cultured for years before being properly stored.

 

One of the key features of the strain that we call NC4 is that it provides phage-like plaques on bacterial agar plates. I think Maurice Sussman is responsible for this as I have earlier isolates of NC4 that are more spready. If you take a wild strain (WS380B, WS576

etc) when you plate it clonally on SM agar, you first see the colony when it is about 1-2cm across as the cells don't eat their way down through the bacteria but instead spread out. This is a disaster for picking out individual colonies as you only get about 8 on a plate before they get mixed up. Maurice understood that for many lab operations you want to be able to pick out clones. NC4 (and the axenic derivatives) give you 100 or so plaques on a plate.

 

My lab has been interested in the slug stage and we found that NC4 and its progeny was not well suited for such studies, so we have done most of our studies on WS380B. The point is that NC4 behaves as an "old" slug from the beginning, and axenic derivatives are even more pronounced. This means that even when you get the slugs to move, they leave very large numbers of cells in their trails (ie they have poor control of their rear). WS380B on the other hand migrates for days and exhibits quite different phases in it's migration. In the beginning it forms the tip so fast that the front gets going before the back is properly formed. This means you have a multicellular organism that is well formed at the front and is being assembled at the back. So the slug gets longer as it moves. Then there is a period when the organisation is sorted out and the rear of the slug

gets pulled up. You then have a lovely slug that moves for several days with only small loss of cells from the rear as it migrates. Finally in old age the control of the rear breaks down and the tail elongates and cells get dropped behind. You can't have such a

precise and clear slug in NC4 strains. We chose to follow the biology and we learned a lot from it.

 

I am not concerned which strain gets sequenced as we know that there is a basic conservation of organisation (you have genetic maps!!), although the plasticity of the chromosomal organisation is pretty scary as Dennis Welker and I found when we were looking at translocations, where large chunks of chromosome got duplicated, chromosomes fused, etc. We know from H.pylori where two isolates have been fully sequenced that there are only(???) a hundred or so genes different between the isolates.

 

Finally just a small comment about the AX2 vs AX3 story. I think these strains are very similar genetically and I wouldn't be surprised if many of the differences being talked about also occur BETWEEN isolates of the same strain (ie the within lab strain variation of AX3 or AX2 may be almost as great as the AX2 vs AX3 variation).

 

The Gerisch approach is the model for anyone starting out. If you follow his protocols you'll be able to be confident that you have the same organism year after year.

 

Bring on the genome as it is going to revolutionise the field.

 

cheers

 

keith

Date: Thu, 4 Nov 1999 22:30:09 -0500

Reply-To: Dicty@listserv.ACNS.NWU.EDU

Sender: owner-Dicty@listserv.ACNS.NWU.EDU

X-Phforward: V2.0@iris

From: David Knecht <knecht@uconnvm.uconn.edu>

To: Dicty@listserv.ACNS.NWU.EDU

Subject: RE: Strain History

I have been out of touch, but I guess I should weigh in on several issues here. First, as to the naming of AX4, I guess I have to admit to being the major culprit. My recollection of events is slightly different from Bill's. When Wolfgang Nellen and I were struggling to get transformation to work, there was a point at which I got cells from him (the Firtel lab,

that is) to see if there was any difference in those cells and ours. In those days, Rick's lab every so often came downstairs to get starter cultures so my assumption was that I was getting back a variant of what we gave them. The cells I received were, as I recollect, called AX3, but behaved very differently from our Loomis lab AX3. I had no knowledge of KAx3 at that time, and so I felt it appropriate to extend the naming technology when we published work with the strain, and so we beget AX4. The difference became particularly obvious when we later made myosin mutants by antisense. Our lab AX3 did not form large multinucleated cells unless you grew them in shaking suspension, while "AX4" made multinucleates on dishes routinely. I have no data to claim that AX4 is not KAX3 and so all the comments made about history are probably correct. That said, I have to also agree with comments of Michael and others, that maintenance of strains is a big problem in the community. We have received numerous strains from other labs with designations of AX3, AX2 etc. that were clearly divergent from the original isolates, and so I agree with the comment that the difference between different lab AX3's is likely to be as great as that between AX2 and AX3. This should scare all of us, since this is a preventable problem and will only cause us increasing problems as technology for examination of phenotypes gets more sophisticated. Anyone who has tried collaborating with a lab that uses different lab stocks I am sure has faced the problems this creates. While I sympathize with Rex's argument that we all have lots of history built up with particular stocks, I think that it would make sense to reconsider this issue as we enter the

age of teh genome sequence. I would not argue that we should all work on AX2 or KAx3, but I would argue that if we say AX2, we should all have the same AX2. Some time ago, I proposed that perhaps we should identify a strain center or repository and have someone distribute to all those in the community who are concerned, a vial of the "real thing" with the intent that we would all maintain Gerisch level of fastidiousness thereafter. Rich could be it for Kax3, and someone in Europe for AX2. I can provide

NC4A2, a strain that we like and use routinely. Obviously, no one is going to recapitulate all their mutants in all new strains, but my experience says that by looking at different parental strains you often learn important information about your gene (witness the complete supression of the lethality of the myosin knockout in KAX3 on plastic). We

see differences in phagocytosis, macropinocytosis, SM plaques, and the phenotype of Rac mutations depending upon the strain in which we look. Some mutants have the same effect in all strains, some have subtle differences, some completely lack comparable phenotype. I consider this a benefit since any phenotype that is conserved, is really clear and important, and one that is strain specific has to be considered more cautiously. However, you might pick up hints that you wouldn't see otherwise by looking in different parentals. Therefore, we routinely make mutants in several different parental background and compare phenotypes. I would also point out that we are not unique as a community in this regard.

I have heard exactly the same comments made about NIH3T3 cells and the variation in lab stocks. Also, I remember the problems created when two labs knocked out yeast clathrin, and one was lethal, and the other not. The advantage they had is that the had the genetics to find out what the gene was that surpressed the clathrin null mutation. We don't, and so I think we have to face this problem head on and deal with it.

 

As to Tom's question about NC4A2, we routinely transform it. In fact, we see little difference in transformation efficiency between the different parental strains that we have used. We are trying out the NC4 transformation ala Harry and Birgit now, but this will obviously not solve all problems, since those of us interested in vegetative cell biology really like axenic growth conditions for some experiments. NC4A2 helps some, but it does not form AX2 like tight plaques on bacteria so it is terrible for SM screening or cloning. One other point to clarify is that this strain does not form NC4 sized fruiting bodies in my hands. It is similar to KAX3/AX4 in size. In fact, I tried several times, and could not isolate a spontaneous axenic strain with NC4 sized fruiting bodies. Perhaps there is a linkage between the pinocytosis necessary for axenic growth and some later signalling event related to the breakup of the mounds.

 

Dave Knecht

 

Date: Fri, 5 Nov 1999 12:00:01 +0000

Reply-To: Dicty@listserv.ACNS.NWU.EDU

Sender: owner-Dicty@listserv.ACNS.NWU.EDU

X-Phforward: V2.0@iris

From: Jeff Williams <j.g.williams@dundee.ac.uk>

To: Dicty@listserv.ACNS.NWU.EDU

Subject: Re: Strain History

 

Bill

 

Thanks for the information and please can we all have your isolate of Ax4,

as I said earlier, the one we have has a staining pattern with ecmB:lacZ

that looks exactly like an ecmA:lacZ stain pattern. I totally agree about

divergence of poorly maintained strains so perhaps what we collectively

need to do is to set up some kind of strain centre/web page - where the

youngest possible isolates of the commonly used strains are held and where

the information about their properties is also held. What is incontestable

is that there are significant differences between the axenic strains

currently in circulation and, without the classical genetics needed to

produce isogenic strains, we have no way other than caution and wide

dissemination of information to collectively protect ourselves.

I remain confused about the duplication but that is best dealt with in one

to one communication.

 

Jeff