돼지 150-kDa insulin-like growth factor complex의 acid-labile subunit(ALS) 유전자의 intron 및 ALS complementary DNA의 3' 비해독 부위 cloning과 생체조직에서의 ALS 유전자 발현 확인
Cloning of An Intron of the Gene Coding for Porcine Acid-Labile Subunit(pALS) of the 150-kDa Insulin-like Growth Factor Complex and the 3' Untranslated Region of pALS
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본 연구의 목적은 다음과 같다: 1) 돼지에서 150-kDa ternary insulin-like growth factor(IGF) complex의 한 구성 요소인 acid-labile subunit (ALS) 유전자 intron의 존재 확인·cloning 및 돼지 ALS(porcine ALS; pALS) complementary DNA(cDNA)의 3' 비해독(untranslated) 부위(3' UT) 증폭·cloning, 2) intron-spanning primer pair를 이용한 reverse transcription-polymerase chain reaction(RT-PCR) 방법에 의한 돼지 조직에서의 ALS 유전자 발현 분포 확인 및 3) 돼지 hepatocyte에서의 ALS 유전자 발현 여부 확인. 돼지 genomic DNA를 template로 하여 PCR 방법으로 예상되는 intron 부위를 증폭하고 plasmid vector에 삽입하여 염기서열을 결정한 결과 타 종의 ALS 유전자에서와 같은 위치에 1,371-base pair(bp)의 pALS intron이 존재함을 확인하였다. 역시 본 연구에서 간에서 추출한 RNA를 주형으로 시작하여 3' rapid amplification of cDNA end(3' RACE) 방법으로 147-bp의 3' UT를 합성하고 그 염기서열을 결정하였다. RT- PCR 결과 간은 물론 조사된 모든 돼지의 내장기관(신장, 폐, 비장)과 자성 생식기관(난소, 난관, 자궁) 및 골격근육에서 ALS 유전자가 발현됨이 밝혀졌다. 또한 돼지 간 조직에 대한 in-situ hybridization 결과 hepatocyte에서 ALS 유전자가 발현됨이 확인되었다. 이상의 결과는 ALS가 혈중 IGF의 저장/조절체로서의 주기능 외에 모세혈관 밖에서도 미지의 기능이 있을 가능성을 시사한다.
Abstract
The aims of this study were as follows: 1) identification and cloning of an intron of the gene coding for porcine acid-labile subunit(pALS), a component of the 150-kDa ternary insulin-like growth factor(IGF) complex, and amplification and cloning of 3' untranslated region(3' UT) of pALS complementary DNA, 2) identification of pALS gene expression in porcine tissues by reverse transcription-polymerase chain reaction using an intron-spanning primer pair and 3) identification of ALS gene expression in porcine hepatocytes. Following nucleotide sequencing after amplification of pALS gene by PCR using genomic DNA as template with a primer pair spanning the expected intron region and insertion of the PCR product into a plasmid vector, a 1,371-base pair(bp) intron of pALS gene has been identified at a site of pALS gene identical to those of other ALS genes of known species. Also identified and sequenced in the present study was a 147-bp 3' UT following 3' rapid amplification of cDNA end(3' RACE) using RNA isolated from the liver as starting template. Results of the RT-PCR have exhibited that pALS gene is expressed not only in the liver but in other internal organs(kidney, lung, spleen), female reproductive organs(ovary, oviduct, uterus) and skeletal muscle. Moreover, in the liver, the ALS gene was found to be expressed in hepatoctytes by in-situ hybridization. These results suggest a possibility that in addition to the known major role as plasma IGF reservoir/regulator, ALS may also have unknown role(s) in the extra-vascular space.
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