Then, we selected the clone for P4H from the genes showing more than 2-fold difference in expression between the fat and muscle tissues (mean values of Cy3/Cy5 ratio for the P4H gene = 2.
The [alpha]-subunit of P4H gene was cloned into pBluescript vector using a cDNA library prepared with a cDNA Library Kit (Invitrogen, USA).
The gene amplification for the [alpha] subunit of P4H was achieved by using PCR with specific primers, P4H-1 and P4H-2 (Table 1).
7 kb full-length sequence of the gene encoding the [alpha]-subunit of P4H was determined using the primers listed in Table 1 (Figure 1A).
The expression levels of the [alpha]-subunit of P4H were determined with the muscular and adipose tissues at each growth stage (weighing 60, 80, and 110 kg) obtained from Sancheong Berkshire pigs (Figure 2).
To clarify the correlation between the expression level of the [alpha]-subunit gene of P4H and pig speciation, the expression level in Yorkshire pigs was analyzed using the same procedure as mentioned earlier.
With respect to the sub-speciation of pigs, it was found that the expression levels of the [alpha]-subunit of P4H were higher in adipose than in muscular tissue.
From the pigs weighing 110 kg, the growth stage at which expression level of the [alpha]-subunit gene of P4H reached the highest point, brain, fat, muscle, bonded rib, heart, and liver tissues were taken for examination of expression level (Figure 5).
We cloned the gene encoding the [alpha]-subunit of P4H from the cDNA library of Sancheong Berkshire pigs and examined differential expression of the [alpha] subunit of P4H in various tissues using microarray analysis (Kim et al.
For the comparison of the cloned [alpha]-subunit of P4H with the human counterpart (Helaakoski, 1989), a complete amino acid sequence was determined.
The level of expression of the [alpha]-subunit of P4H was significantly higher when the pig weight reached 110 kg, indicating that the collagen content would be higher at this growth stage.
In this respect, it is considered that the [alpha]-subunit of P4H plays an important role in activating the P4H holoenzyme.