Materials and Methods
PFGE and southern hybridization
Chromosomal DNA was prepared essentially as described before (Smith et al, 1987). 1.6 × 108 cells
were collected from the log-phase culture, washed with 50 mM EDTA twice, and suspended in 1 ml of
CSE1 solution [20 mM citrate phosphate (pH 5.6), 1.0 M sorbitol, 50 mM EDTA] supplemented with
Zymolyase 20T (Seikagaku) and Lysing enzyme (Sigma) at the final concentration of 0.25 mg/ml each.
After incubation at 30°C for 30–60 min with rotation, the spheroplasts were collected by centrifugation at
500 rpm for 15 min (MX-201 micro centrifuge, TOMY), washed with 0.5 ml of CSE2 solution [20 mM
citrate phosphate (pH 5.6), 0.9 M sorbitol, and 50 mM EDTA], and resuspended with 200 µl of CSE2.
After addition of 200 µl of 1.6% low-melting agarose LM (Nacalai tesque) solution in H2O that was
pre-incubated at 50°C, the spheroplast suspension was transferred into molds (Bio-Rad) to make four
agarose plugs.
The four plugs were incubated at 60°C for 2 h in 2 ml of ES solution [0.25 M EDTA, 1%
SDS], and then incubated at 50°C for 24 h in 2 ml of ESP solution [0.5 M EDTA, 1.0% N-lauroyl
sarcosine (Sigma), 1.0 mg/ml Proteinase K (Merck)].
The plugs were washed with TE10:1 [10 mM
TrisHCl (pH 8.0), 1 mM EDTA] three times and maintained at 4°C.
Electrophoresis was carried out
using Certified Megabase agarose gel and CHEF-DRII system (Bio-Rad) under the condition indicated in
the figure legends.
After treatment of the gel with 0.25 M HCl for 1 h, with 0.6 M NaCl and 0.2 M
NaOH for 30 min twice, and with 50 mM sodium phosphate (pH 6.5) for 1 h, DNA was transferred to a
Nytran nylon membrane (Schleicher & Schuell).
In the case where chromosomal DNA was digested with the restriction enzymes, 6.25 × 10 8
cells were collected, washed with TE10:25 [10 mM TrisHCl (pH 8.0), 25 mM EDTA] and then with SP1
buffer [1.2 M sorbitol, 50 mM citrate phosphate (pH 5.6), 40 mM EDTA (pH 8.0)], and stored at –80°C
before use.
The cells were resuspended with 1.5 ml of SP1 buffer containing Lyticase (Sigma) at 3.5
mg/ml and incubated at 37°C for 30–60 min.
The spheroplasts were collected by centrifugation at 2,000
rpm for 3 min (GRX-250 centrifuge, TOMY) and suspended with 150 µl of SP1.
After addition of 200
µl of 42°C pre-incubated low-melting agarose LM, the spheroplasts were transferred into molds.
The
plug was incubated overnight at 50°C in 5 ml of DB buffer [1.0 mg/ml Proteinase K (Merck), 1.0%
N-lauroyl sarcosine (Sigma), 25 mM EDTA], and then stored at 4°C in TE10:50 [10 mM TrisHCl (pH
8.0), 25 mM EDTA].
The plug was treated with 10–20 U of a restriction enzyme at 37C for 3 h.
plug was melted by incubation at 70C for 10 min.
The
After addition of 2 µl of 0.5 mg/ml RNase A
(Sigma) and 2 U of -agarose I (New England Biolabs), the solution was incubated at 42°C for 1 h,
followed by the addition of 20 U of restriction enzyme and overnight incubation at 37°C.
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After
centrifugation at 13,000 rpm for 10 min (MX-201, TOMY), DNA in the supernatant was alcohol
precipitated.
0.8 and 0.7% agarose gels were used for the BmgBI and XhoI cases, respectively.
Instead
of HCl treatment, the agarose gel was irradiated with UV light (300 mJ).
Plasmids
A genomic region that contains the rad51 gene was amplified using rad51-5’ and rad51-3’ primers,
digested with BglII and HindIII.
The resulting 2.2-kb restriction fragment was introduced between the
BamHI and HindIII sites of pBluescript II KS+, creating pTN466.
A 1.5-kb PmeI-BamHI fragment
containing the kan gene from pFA6a-kanMX6 was introduced between the BstI and BamHI sites of
pTN466, creating pTN471.
A 1.4-kb region near the gpi8 gene was amplified using ChLR-F and
ChLR-R primers and digested with EcoRI and HaeIII.
The resulting 1.3-kb EcoRI-HaeIII fragment was
introduced between the EcoRI and EcoRV sites of pBluescript II SK+, creating pTN672, and used to
prepare probe B.
pTN445 harboring a 3.0-kb XbaI-BamHI fragment containing the ura4+ gene on SK+
was digested with HindIII and self-ligated, creating pTN753.
pTN753 was used to prepare probe E.
A 0.8-kb XbaI-HindIII fragment from
A 1.8-kb fragment near the chk1+ gene was amplified using
III_1062F and III_1062R primers and digested with ClaI and HindIII.
The resulting 0.7-kb fragment
was introduced between the ClaI and HindIII sites of SK+, creating pTN754, and used to prepare probe F.
A 2.2-kb region near spcc4B3.18 was amplified using cen3R-F and cen3R-R primers and digested with
EcoRI.
The resulting 0.6-kb EcoRI fragment was cloned into the EcoRI site of SK+, creating pTN755,
and used to prepare probe A.
A 1.0-kb region near the ade5+ gene was amplified using the ChIIIR-F and
ChIIIR-R primers and digested with EcoRI and XbaI.
The resulting 0.8-kb EcoRI-XbaI fragment was
cloned between the EcoRI and XbaI sites of SK+, creating pTN756, and used to prepare probe D.
A
2.2-kb HindIII fragment from pREP1 (Maundrell, 1993) was used to prepare the LEU2 probe.
A
10.4-kb HindIII fragment from YIp10.4 (Toda et al, 1984) was used to prepare the rDNA probe.
A
3.6-kb region near the ams2+ gene was amplified using the ams2-F and ams2-R primers and digested with
XbaI and ClaI.
The resulting 3.1-kb XbaI-ClaI fragment was cloned between the XbaI and ClaI sites of
KS+, creating pTN861.
A 2.1-kb XbaI-PstI fragment from pTN861 was used to prepare probe C.
A
1.8-kb HindIII fragment containing the ura4+ gene from pTN854 was used to prepare the ura4 probe.
A
1.9-kb DraI fragment containing the ade6+ gene from pTN437 was used to prepare the ade6 probe.
References
Maundrell K (1993) Thiamine-repressible expression vectors pREP and pRIP for fission yeast. Gene
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123(1): 127-130
Smith CL, Matsumoto T, Niwa O, Klco S, Fan JB, Yanagida M, Cantor CR (1987) An electrophoretic
karyotype for Schizosaccharomyces pombe by pulsed field gel electrophoresis. Nucleic Acids Res
15(11): 4481-4489
Toda T, Nakaseko Y, Niwa O, Yanagida M (1984) Mapping of rRNA genes by integration of hybrid
plasmids in Schizosaccharomyces pombe. Curr Genet 8: 93-97
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