ABSTRACT : The mammalian cellular retinoic acid-binding proteins, CRABP-I and CRABP-II, bind retinoic acid which acts as an inducer of differentiation in several biological systems.
Retinoic acid (RA) is bound to two intracellular proteins, the cellular retinoic acid-binding proteins type I and II (CRABP-I and CRABP-II) that are highly conserved among several animal species (Li and Norris, 1996; Miano and Berk, 2000).
To clone bovine CRABP-II, 1 [micro]l of synthesized cDNA from bovine sirloin tissue was added to a sterile 0.2-ml tube containing 5 [micro]l of 10xPCR buffer, 4 [micro]l of 10 mM dNTP mixture, 1 [micro]l of forward primer (20 pmol/[micro]l , TaKaRa, Korea), 1 [micro]l of reverse primer (20 pmol/[micro]l , TaKaRa, Korea) and 0.5 [micro]l of EX Taq DNA polymerase (5 U/[micro]l , TaKaRa, Japan) and the volume was adjusted to 50 [micro]l with distilled water.
To determine the tissue expression of bovine CRABP-II mRNA, total RNA was extracted from heart, liver, lung, colon, small intestine, spleen, rib meat, sirloin and adipose tissues of a 12-month-old bull.
Comparative real-time PCR was performed using a Roter Gene 2000 system (Cobett Research, Australia) and a QuantiTect SYBR Green RT-PCR kit (QIAGEN, Germany) according to the manufacturers' instructions, with the following primer pairs: CRABP-I sense, 5'-AAGGCTTTGAGGAGGAGACC-3'; CRABP-I antisense, 5'-TCAGGATGAGTTCGTCGTTG-3'; CRABP-II sense, 5'-AAACAGGAGGGGGACACTTT-3'; CRABP-II antisense, 5'-CCCATTTCACCAGGCTCTTA-3'; 18S rRNA sense, 5'-CCCGAAGCGTTTACTTTGAA-3'; and 18S rRNA antisense, 5'-CCCTCTTAATCATGGCCTCA-3'.
Cloning and characterization of bovine CRABP-II cDNA
A cDNA encoding a bovine CRABP-II was isolated from bovine sirloin tissue by RT-PCR using degenerative primers.
The mRNA expression levels of CRABP-II were undetectable by Northern blot analysis using various bovine tissues (data not shown).
In contrast, bovine CRABP-II transcripts were detected in undifferentiated intramuscular fibroblast-like cells and their levels on day 15 had been induced approximately seven-fold compared to day 0 (Figure 4C).