This part of the Forsburg Lab website contains technical information of use to people who study S. pombe. Visit our home page for a directory to this pombe site, or the list of frequently asked questions for fast answers to common questions. If you want to browse practical information about working with fission yeast, you're in the right place.
Commonly used selectable markers.A note about nomenclature. A summary of promoter activity and expression systemsInfo on primer design for amplification of open reading frames. How to construct plasmids for cross-complementation experiementsThe almanac of useful constants and numerical values for pombeade6 mutant alleles that may be hanging out in your strains
The "wildtype" 972: where did it come from?
Restriction site usage in S. pombe genome.
Protocols for pombe, including flow cytometry, colony PCR, working with diploids, disruptions and integrations, DAPI staining, and plasmid shuffle. Also includes links to other protocol pages.
Other pages on our site: follow these links to find
Frequently asked questions about working with pombe
PombeWeb, including fission yeast lab home pages, faculty listings, meetings, genomics, and all the pombe-related links we can find.
Community news, including newsbites, committee info, and postdoc ads
An index of fission yeast plasmids, including general plasmid information, sequences, and maps (the vector database).
A list of useful technical references for fission yeast molecular genetics.
Genome project information for fission yeast
Sequence analysis sites including Sanger, the Blast servers and analysis tools
Recipes for fission yeast media
Drugs for fission yeast, including selection/counterselection and DNA damaging agents
Want to know the equivalent S. cerevisiae genes for S. pombe cell cycle genes? Visit our new gene conversion table.
Where to get strains and plasmids.
A codon usage table for fission yeast.
Other sites (see also our protocols section)
This Yeast genetics course provides a well-illustrated site describing genetic methods in both common laboratory yeast species
Can't find what you need? Try posting a message on the Bionet newsgroup Yeast.
Genetic mapping data of S. pombe and explanatory text, from Peter Munz. These were used to construct the map in the Academic Press pombe book
General nomenclature rules: Fission yeast gene names are generally expressed as a three letter, italic name followed by a number, and a plus for wild type (cdc19+; the plus should be superscripted in normal text). In mutants, the plus is replaced by an allele designation (cdc19-P1). Allele designations vary widely in format; some are letters, some numbers, some a mix.
Most investigators indicate deletions with a 
(delta). For example, a disruption of your favorite gene yfg1+ with ura4+ is written yfg1::ura4+ in a proper strain table, although people often use yfg1 as short hand in text. For deletion alleles constructed without an insertion, some use an allele designation beginning with D; e.g, ura4-D18 is a deletion of ura4+. However, you cannot be sure that a "D" in an allele designation is always a deletion, so use caution and read the literature. It is strongly encouraged that deletion alleles be given a unique allele designation, e.g., yfg1-D1::ura4+. In several cases, the same gene has been disrupted in different laboratories, and the phenotypes vary according to the precise construct. Without a unique identifier for a given lab's allele, this has led to considerable confusion in the literature.
The :: (double colon) is used to indicate an insertion into the genome. It need not correspond to a deletion/disruption, for example, marking a locus by integrating a marker downstream would be indicated yfg1+::leu1+ or yfg1+::pAB123. Generally, what comes before the :: is the locus, and you need to indicate whether it is wild type or mutant; what comes after the :: is what was integrated, and you need to identify it as well.
Proteins are indicated by roman text, often followed by an appended p (eg, Cdc19p). The p is particularly useful for non-yeast people who might be unfamiliar with the conventions of roman versus italic type, but it is viewed as optional. Just to complicate life further, budding yeast nomenclature is different: the wildtype gene names are in italic capitals (LEU2), mutants (unless dominant) are in lower case (leu2-3) and protein names are in roman text (LEU2p or Leu2p).
The common promoters in fission yeast are adh1+ (constitutive high expression), fbp1+ (carbon source responsive), a tetracycline-repressible system based on the CaMV promoter, and the nmt1+ (no message in thiamine) promoter, which is the most frequently used.
There are three versions of nmt1+ promoter: the full strength promoter, and two attenuated versions that have reduced activity both in repressed and induced conditions (indicated below as nmt* and nmt**; see references). Several different polylinkers are available in the REP/RIP series of nmt vectors (see vector database). The concentration of thiamine can be adjusted for partial activation. Full induction: no thiamine. Full repression: 15uM thiamine (5ug/ml). Partial induction (described in this reference): 0.05uM thiamine (0.016ug/ml).
The nmt1 promoter does not switch off completely, and the ability to construct a "shut-off" plasmid depends very much on the protein being expressed and the sensitivity of the cell to dosage of that particular protein. Many genes expressed under nmt1 control are able to complement even in the presence of thiamine in the weakest promoter, but there are also numerous examples of genes that can be successfully shut off to generate a null phenotype. Thus, the utility of this promoter for plasmid shut-off experiments must be determined empirically for each gene.
A comparison of promoter activity was published in Forsburg, (1993). Nucl. Acids Res. 21, 2955-2956. The data are summarized as follows (measuring units of beta galactosidase activity produced by lacZ fusions). The first three are constitutive, although the CaMV promoter can be rendered inducible by coexpression of a tet repressor (see vectors page).
From Juerg Bahler: Web-interface scripts that automatically design primers for PCR-based gene targeting. (Click link for "software"). This will automatically suggest primer sequences for gene deletion, tagging, and/or regulatable expression based on gene name and plasmid information that you specify.
We are asked periodically whether S. cerevisiae plasmids will function in S. pombe. The answer is, don't count on it. Fission yeast replication origins, promoters, and splicing mechanisms are all quite different from those in budding yeast. Thus, expression of marker genes is likely to be poor to non-existant. (The notable exception is the ScLEU2 marker, which works reasonably to complement Spleu1, at least in high copy.) The promoter of your favorite cerevisiae gene probably won't work in fission yeast, and it won't be spliced correctly. And in the absence of a pombe-specific replication origin, the plasmid will transform inefficiently, if at all, and will be prone to re-arrangement.
So how to do cross complementation experiments? the answer is, with a cDNA (or a clone without introns), in a plasmid built for pombe --including marker, origin, and promoter driving the cDNA. That way, you can at least be confident your clone expresses and draw a meaningful conclusion from your experiment! Note this also is true for genes from other organisms. Plug any cDNAs into a pombe expression vector and you can try to complement a conditional mutant strain by standard transformation. If the only pombe mutant you have is a lethal disruption, you can do the experiment as described on our diploids page.
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For comparison of genome sizes from other organisms, see this page from Zac Cande's lab.
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People doing flow cytometry on exponentially growing S. pombe are puzzled that there is only one peak, corresponding to 2C DNA content. Fission yeast DOES have a G1 phase, but it is short (about 10% of the cell cycle). And, the timing of cytokinesis and the timing of the nuclear division cycle are somewhat out of synch. This means that after nuclear division (mitosis) occurs, the nuclei go through G1 and enter S phase before the cells complete cytokinesis. As far as a flow cytometer is concerned, a binucleate cell with two 1C (G1) nuclei looks the same as a uninucleate cell with a single 2C (G2) nucleus.
If you have a javascript-enabled browser, move your mouse over the image at right, and watch a simple graphic of dividing pombe. See how the nuclei are divided prior to cytokinesis?
For a FACS profile of normal, wild type cells, with explanations, click here. Use your Back button to return.
Joel Huberman has a quantitative microscopy method that correlates nuclear morphology with DNA content, and provides further demonstration of this fission yeast feature (not a bug!).
For general advice about yeast genetics and strategies for designing screens, see this recent review by Susan.
For more detail, we provide several protocols here on our site:
A number of other sites also offer useful protocols:
For general molecular and cell biology protocols and servers, try these index sites:
We have had success generating ts alleles using plasmid shuffle strategies in pombe. Although written for cerevisiae, this protocol for isolation of ts mutants more or less explains how it's done. Our method: we isolate a haploid containing a disruption of our favorite gene yfg1+, covered by a ura4+-marked (or tk-marked) plasmid containing the wild type gene (call it plasmid 1). We use hydroxylamine for in vitro mutagenesis of another yfg1+ plasmid, marked with something else (plasmid 2). We transform plasmid 2 into the starting haploid, use plasmid shuffle with FOA or FUDR selection against plasmid 1, and screen for ts mutants that contain plasmid 2 and lack plasmid 1. Upon isolation and retesting of the plasmid to confirm the temperature-sensitive phenotype, we integrate back into the chromosomal locus.
Check out this review for general yeast genetics information.
There are numerous ade6 alleles used for various reasons in various strains, which gets confusing for everyone. Here is a guide to some of them. We would like to be inclusive, so please send us references and info for any other alleles you know about! Enormous thanks to Juerg Kohli, who corrected what we had and provided much more. Also, thanks to John Armstrong, Ramsey MacFarlane, Greg Freyer, Jun Gao, Dan Pankratz and Katja Ludin for providing additional information.
| Alleles of ade6+ | ||||
|---|---|---|---|---|
| allele | nt change (if A of ATG is 1) | aa change | comments | References |
| M26 | G135T | G46 to stop | dark pink on low ade. UGA codon, can suppress with sup3/9/12. Hyper-rec. Adjacent codon to M375 | Szankasi et al 1988 |
| 51 | C1266T | H423Y | Schär et al 1993 | |
| 52 | T955C | L319S | Pale pink on low ade. Will complement with M210 (D. Pankratz). | Schär et al 1993 |
| 149 | C1180T | S394L | Schär and Kohli 1993 | |
| M210 | C1466T | P489L | dark pink on low ade. Will complement with M216. Although most agree on this mutation, Greg Freyer reports his as a G1470A lesion, making a G491S mutation. In all cases, it destroys the Xho site. | Macfarlane and Wahls labs |
| M216 | G46A | G16D | pale pink on low ade. Will complement with M210 and 149 | Szankasi et al 1988 |
| M375 | G132T | G45 to stop | dark pink on low ade. UGA codon; suppressed by sup3/9/12 Adjacent codon to M26. | Szankasi et al 1988 |
| M387 | G1270C | R424P | G to C marker effect | Schär et al 1993 |
| 406 | G2A | M1I | Schär and Kohli 1993 | |
| 421 | G1176A | G393S | Schär and Kohli 1993 | |
| 424 | C1261T | S421L | Schär and Kohli 1993 | |
| 424 | T1222-T | Frameshift | Schär and Kohli 1993 | |
| 469 =L469 | C1467T | R490 to stop | dark pink on low ade. UGA codon; Can suppress with sup3/9/12. Destroys Xho site | Szankasi et al 1988 |
| 485 | C1244G | Y415 to stop | G to C marker effect. UAG codon (not suppressible) | Schär and Kohli 1993 |
| 555 | C124T | ? | Ludin, MS thesis | |
| 687 | T341 +T | Frameshift (114) | Schär and Kohli 1993 | |
| 704 | T645A | C215 to stop | v. dark pink on low ade. Can suppress with sup3. (Note conflict: reported by Schär and Kohli 1993 as C845A?; C282 to stop. It is possible there are two different UGA nonsense alleles both suppressible by sup3-5, and both are called 704) | Park JM, Intine RV, Maraia RJ. Gene Expr. 2007;14(2):71-81. |
| 706 | C48T | R51 to stop | v. dark pink on low ade. UGA codon. Can suppress with sup3/9/12. | Grimm et al, 1994 |
| A1943 | G1068A | G357R | Schär and Kohli 1993 | |
| T1994 | C1119T | Q374 to stop | UAG codon | Schär and Kohli 1993 |
| A2097 | T1222A | L408 to stop | UAA codon | Schär and Kohli 1993 |
| A2196 | T1321A | L441 to stop | UAG codon | Schär and Kohli 1993 |
| A2244 | T1369A | M487K | Schär and Kohli 1993 | |
| T2291 | G1416T | G473 to stop | UGA codon | Schär and Kohli 1993 |
| N/N | internal deletion | v. dark pink on low ade. Minigene. | ? | |
This information comes from John Armstrong and Paul Young during a recent conversation on the pombelist mailing list
John notes:
"the standard pombe strains derived from Leupold's 972 are not connected to Lindner's original isolate, but apparently originate in
some rancid grape juice from Montpelier, Switzerland, in the 1920's.
Some of this is described in the chapter by Munz et al in Molecular
Biology of the Fission Yeast. So they have never been to East Africa and
never used to make pombe. Secondly, NCYC keep a strain of pombe called NCYC132 which some early workers such as Mitchison and Nurse described. It's very different from 972 strains and a bit messy to work on. .....The NCYC database describes neither a year of deposition nor a depositor. However I suspect its origins are separate from 972, and they might even be in East Africa."
However, Juerg Kohli notes (citing Urs Leupold in The Early days of Yeast Genetics book) "The standard S. pombe strains were isolated by Urs Leupold in 1946 and 1947 from a culture that he obtained from the yeast collection in Delft, The Netherlands. It was deposited there by A. Osterwalder under the name S. pombe var liquefaciens, after he isolated it in 1924 from French wine (most probably rancid) at the Federal Experimental Station of Vini- and Horticulture in Wdenswil, Switzerland. The culture used by Urs Leupold contained (besides others) cells with the mating types h90 (strain 968), h- (strain 972), and h+ (strain 975)."
Paul adds about the mysterious 132::
"At least one difference for 132 is that when challenged with minus nitrogen, 132 does not accelerate mitosis within the largest G2 cohort but rather divides down progressively (Mitchison, pers.com.). As for other isolates I see a recent paper in Pubmed noting that it is
common in Kambucha mix and it has also been reported as a (the?) major yeast in cactus beer (pulqa sp?) in Mexico. "
This table was generated by A. John Callegari at Sloan Kettering (callegaa AT mskcc.org). Send questions or comments to John.
| Restriction site usage in S. pombe genome | |||
|---|---|---|---|
| Enzyme | Sites in Pombe Genome | Random DNA Expect | Average fragment size |
| PvuII | 2,142 | 3,076 | 5,882 |
| PmlI | 684 | 3,076 | 18,421 |
| AluI | 47,242 | 49,218 | 266 |
| RsaI | 29,655 | 49,218 | 424 |
| BsaBI | 3,103 | 3,076 | 4060 |
| BsrBI | 1,328 | 3,076 | 9487 |
| DraI | 15,932 | 3,076 | 790 |
| EcoRV | 4,965 | 3,076 | 2537 |
| HpaI | 1,801 | 3,076 | 6,996 |
| NaeI | 365 | 3,076 | 34,520 |
| StuI | 911 | 3,076 | 13,830 |
| AscI | 9 | 192 | 1,400,000 |
| NotI | 13 | 192 | 969,230 |
| BamHI | 1,460 | 3,076 | 8,630 |
| BglII | 2,042 | 3,076 | 6,170 |
| EcoRI | 4,180 | 3,076 | 3,014 |
| HindIII | 6,457 | 3,076 | 1,951 |
| MluI | 823 | 3,076 | 15,309 |
| PvuI | 1,012 | 3,076 | 12,450 |
| KpnI | 1,165 | 3,076 | 10,815 |
Stats are based on the three chromosomes downloaded from genbank in August, 2006. There are 12.6 megabases in this sequence, suggesting that about 8.7% of the genome is missing (mostly likely repetitive DNA).Therefore, these stats must be viewed as approximations.
DAPI staining fixed cells: we have used three methods. These can be used on live cells (less efficient), or on fixed cells. If not treating for immunofluorescence, we find that ethanol fixation (as done for FACS) works quite well, as does heat fixation performed by putting a small aliquot of culture on a slide and exposing it briefly to a hot plate.
A 10x DAPI mounting stock may be prepared and aliquoted. This is 10µg/ml DAPI, 10mg/ml p-phenylenediamine . (The DAPI stock is 1mg/ml in DMSO. ) Take a 20µl aliquot and add 180µl 100% glycerol . This gives a 1X working stock (1µg/ml DAPI, 1mg/ml p-phenylenediamine which acts as anti-fade, 90% glycerol) which should be kept at -20°C in the dark.
You can use DAPI, but it doesn't get into live cells quite as well. Hoechst 33442 has been recommended as a more effective live cell nucleic acid stain. When we get the conditions and concentrations, we will add them here.
© S. L. Forsburg .
