The murine gene Cdkn1b (p27
) maps to distal Chromosome 6 and
is excluded as Pas1
Christopher J. Kemp, Kyung-Hoon Kim, Jeannette Philipp
Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N., Seattle, Washington 98109, USA
Received: 13 August 1999 / Accepted: 11 January 2000
Activation of cyclin-dependent kinase-2 (cdk2) by cyclin E is a
key event in driving cells from G1 into S phase of the cell cycle.
Cdkn1b, also known as p27
or p27, belongs to the Kip/Cip
family of cdk inhibitors and responds to extracellular signals such
as TGFB and cell contact by binding to cyclin E/cdk2 complexes
(Polyak et al. 1994). This binding inhibits cdk2 activation and
thereby blocks cells from entering S phase. Cdkn1b also binds to
cyclin D/cdk4/6 and cyclin A/cdk2 complexes. Quiescent cells
generally express higher levels of Cdkn1b than proliferating cells,
and enforced expression of Cdkn1b inhibits cell proliferation.
Mice that are deficient in the Cdkn1b gene owing to gene knockout
are larger than control littermates, confirming a role for Cdkn1b in
growth control (Fero et al. 1996). These mice are also predisposed
to chemical and radiation-induced tumorigenesis in multiple tis-
sues, indicating that Cdkn1b is a tumor suppressor gene (Fero et al.
1998). To further understand the role Cdkn1b plays in murine
cancer, we determined its chromosomal map position.
The p27 genomic sequence was originally obtained from a p27
clone that was isolated from a murine T-cell genomic library using
a labeled p27 cDNA as probe (Kwon et al. 1996). The nucleotide
sequence accession No. in GenBank is U51681 (Kwon et al. 1996).
The cloned genomic sequence was a 5.6-kb BamHI fragment con-
taining the complete p27 coding sequence and ∼2 kb sequence 5Ј
to the transcription start site. The p27 genomic locus consists of
two coding and one noncoding exon, and the protein is 197 amino
In searching for a polymorphism at the p27 locus, we noticed
two closely spaced CA repeats at ∼1800 bp 5Ј to the p27 tran-
scription initiation site (Kwon et al. 1996). We designed the fol-
lowing primers: 5Ј-GTT ACT TTT GAG TGC AGG AG-3Ј and
5Ј-TTT CTT AGC CAC ATC TTT GC-3Ј to amplify both CA
repeat regions together. We used standard PCR conditions with 3.0
, 55°C annealing temperature, and 40 cycles and ana-
lyzed the products on 0.5% agarose, 2% Nu Sieve agarose (FMC
bioproducts) gels, and ethidium bromide staining. We observed the
following size polymorphisms in different inbred strains: 129/SvJ:
95 bp; C57BL/6J: 165 bp; M. spretus: 100 bp; C3H/HeJ: 90 bp;
and NIH/Ola: 85 bp. Sizes given are approximate.
The chromosomal location of Cdkn1b was determined by using
The Jackson Laboratory interspecific backcross panels: (C57BL/6J
× M. spretus)F
× C57BL/6J, called Jackson BSB, and (C57BL/
6JEi × SPRET/Ei)F
× SPRET/Ei, called Jackson BSS (Rowe et al.
1994). The C57BL/6J Cdkn1b allele was followed as a 165-bp
PCR fragment, and the M. spretus allele as a 100-bp fragment. In
the 94 DNA samples from the BSB panel, we observed only one
recombinant between Cdkn1b and D6Mit220 and one recombinant
between Cdkn1b and D6Mit25. In the 94 samples from the BSS
panel we observed no recombinants between Cdkn1b and
D6Mit220. Thus, the murine Cdkn1b gene maps to distal Chro-
mosome (Chr) 6 as shown in Fig. 1.
The human p27/Kip1 gene maps to Chr 12p13 (Pietenpol et al.
1995). There is a large region of conserved gene order between
human 12p12-12p13 and murine Chr 6 (see Fig. 1), and thus
Cdkn1b/p27/Kip1 is added to the list of genes in this grouping.
In the BSS panel, there were only five recombinants between
Cdkn1b and Kras2, indicating linkage between the tumor suppres-
sor gene Cdkn1b and the oncogene Kras2. A murine pulmonary
adenoma susceptibility locus, Pas1, has also been mapped to an
18-cM region of distal chromosome 6 (Gariboldi et al. 1993),
which includes both Kras2 and Cdkn1b (Fig. 1). The A/J and
BALB/c strains carry the susceptible allele of Pas1, and these mice
are of high and intermediate predisposition to lung tumor devel-
opment, respectively (Manenti et al. 1997). C57BL/6J, C3H/He,
and M. spretus mice carry the resistant Pas1 allele, and these
strains are resistant to lung tumorigenesis (Manenti et al. 1995).
The Kras2 oncogene is frequently mutated in murine lung tumors,
is polymorphic in some of the strains described above, and is a
candidate gene for Pas1 (Chen et al. 1994; Ryan et al. 1987),
although this has been questioned (Manenti et al. 1995). As
Cdkn1b is a lung tumor suppressor gene in mice (Fero et al. 1998)
and maps within the Pas1 interval, it is also a candidate for Pas1.
Therefore, we sequenced the entire coding region of Cdkn1b in
genomic DNA samples prepared from three resistance strains
C57BL/6J, M. spretus, and C3H/HeJ, two susceptible strains A/J
and BALB/c, as well as from 129/Sv and NIH/Ola (from Harlan
Olac, UK) strains. Genomic DNA from C57BL/6J, C3H/HeJ, A/J,
129/SvJ, BALB/cJ, and SPRET/Ei was obtained from The Jackson
Laboratory. We also prepared genomic DNA from tail snips from
C57BL/6J, C3H/HeJ, A/J, 129/SvJ, BALB/cJ, M. spretus, and
NIH/Ola mice from our own colony, using a QIAamp DNA mini
A 655-bp PCR product including exon 1 of Cdkn1b was am-
plified with standard PCR (3.0 m
and 55°C annealing
temperature with 40 cycles), with 5Ј-AAG AGG GTT TTG CGC
TCC AT-3Ј and 5Ј-AGT AAA GGG GCG CTT ACG GA-3Ј prim-
ers. We used the internal primer 5Ј-TTC CGG AGA GAG GCG
AGG CG-3Ј to sequence exon 1, using PE biosystem Dye-
Terminator® as well as Big-Dye® cycle sequencing.
Exon 2 of Cdkn1b was amplified by PCR with 5Ј-GTC AGC
CAT TGT TGT CGA TAA TA-3Ј and 5Ј-TGA TCA AGG AAA
GTA ATT CCT C-3Ј primers. The 328-bp PCR product was li-
gated into the lacZ gene of pCR2.1 (Original TA cloning kit from
Invitrogen), followed by transformation into INV␣F cells (One
Shot®). Transformed cells were grown overnight on an agar plate,
white colonies were picked, and plasmids were isolated and ana-
lyzed by restriction mapping. The inserts containing exon 2 were
sequenced with external M13 reverse primer 5Ј-CAG GAA ACA
GCT ATG ACC-3Ј with PE biosystem Big-Dye® cycle sequenc-
Correspondence to: C.J. Kemp; e-mail: email@example.com
Mammalian Genome 11, 402–404 (2000).
© Springer-Verlag New York Inc. 2000