Determination of reiteration frequencies of
Wpkci and chPKCI in the chicken genome.
(A) The 140-kb insert containing Wpkci genes was
obtained from the BAC clone 216G1 by digestion with NotI
and further digested with PstI at six different
concentrations. The digests were separated by agarose gel
electrophoresis, stained with ethidium bromide (left panel), and
subjected to Southern blot hybridization with the
32P-labeled insert of the cDNA clone fst5.2-5 (right panel;
see Figure 2A), which showed signals corresponding to multiples of the
5.6-kb repeating unit. (B) The insert of BAC clone 224D8 containing the
chPKCI gene sequence was obtained by NotI
digestion, further digested with BamHI at eight
different concentrations, and subjected to Southern blot hybridization
with the 32P-labeled cDNA fragment (nucleotide positions
30–410) of chPKCI. (C) Different amounts of
EcoRV-digested genomic DNA of the female chicken (upper
panel) and EcoRV-digested genomic DNA of the male
chicken (3 μg each) mixed with different amounts of the linearized
cDNA clone p5fm2 (3.83 kb) (lower panel; see also Figure 2A) were
subjected to agarose gel electrophoresis and Southern blot
hybridization with the 32P-labeled insert of p5fm2.
Comparing the slopes of signal intensities for the former and the
latter samples, 1 μg of the female genomic DNA and 72 pg of p5fm2
gave the same signal intensity. (D) The BamHI-digested
genomic DNA of the male chicken (3 μg each) and different amounts of
the HindIII-digested, linearized cDNA clone pchPKCI-3
were subjected to electrophoresis and Southern blot hybridization with
the 32P-labeled subfragment (nucleotide positions 30–410)
of pchPKCI-3. Comparing the mean signal intensity of the former and the
slope of signal intensities of the latter samples, 3 μg of the male
genomic DNA and 12 pg of pchPKCI gave the same signal intensity.