The mcm4–
c70, mcm4–c84, and mcm4–c106 mutants were created by yeast transformation
with SpeI-digested pTN565, and XbaI-digested pTN558 and pTN559,
respectively. These plasmids were constructed as follows. Introducing the
BglII-EcoRI restriction sites immediately after the mcm4 stop codon, a 1.3-kb
EcoRV-BamHI fragment containing the Mcm4 C-terminal region was amplified
by PCR and then cloned between the EcoRV and BamHI of pBluescript II
KS_, creating pTN528.A1.5-kb BglII-EcoRI fragment containing kanMX6 from
pFA6-kanMX6 (41) was cloned into the BglII and EcoRI sites of pTN528,
creating pTN529. The reverse primer (5_-AACAGCTATGACCATG) was paired
with the forward mutagenesis primer mcm4-cd70 [5_-CGAGATCTTAGACCATATCTTCAGGTACCAAAG
(the underlined portion represents the BglII site,
and the italicized portion represents the stop codon that was introduced)],
mcm4–cd84 (5_-CGAGATCTTAAATTAGGTCAAGAGAAATCTTTCC), or mcm4–
cd106 (5_-CGAGATCTTACAAGCGAGCAGCTTCAAGAACATC) in PCRs using
pTN529 as a template. The PCR products were digested with XhoI and BglII,
and the resulting 1.1-kb XhoI-BglII fragments were introduced into the XhoI
and BglII sites of pTN529 to create pTN565, pTN558, and pTN559, respectively.
The absence of any additional mutations in the PCR fragment was confirmed
by DNA sequencing. A 4.3-kb region of yeast genomic DNA that contains
SPCC584.10c was amplified by using a pair of primers (5_-CGCAAGGGCGTCGTTACCCATGG
and 5_-CTCAACTACCGGCTAAGGTTGGC), digested with HindII
and NheI, and the resulting 3.6-kb HindIII-NheI fragment was introduced into
the HindIII and XbaI sites of pBluescript II KS_, creating pTN807.