백서 간장의 ATP-citrate lyase에 대한 cDNA cloning과 insulin에 의한 APT-citrate lyase 생합성 조절 기전

Abstract

[한글]

백서 간장세포에서 insulin에 의한 ATP-citrate lyase (ACL)의 생합성 조절 기전을 밝히기 위하여 백서 간장세포의 cDNA가 삽입된 λgtll library (Kim등, 1989)로부터 순수정제된 anti-ACL IgG를 사용하여 ACL cDNA가 삽입된 양성 clone 25개를 선별 분리하여 분리된 각 clone을 각각 λACLI-λACL25로 표기하고 각 clone으로부터 phage DNA를 분리하여 삽입된 cDNA 크기를 측정하였다.

그 결과 λACLI은 0.7 kb, λACL3과 λACL4는 1.0 kb와 0.5 kb (2개), λACLS는 0.6 kb, λACL8은 1.0 kb, λACL9는 0.7 kb, λACLll은 0.95 kb와 0.5 kb (2개), λACL12는 0.7 kb, λACL15는 1.2 kb와 1.0 kb (2개), λACL17은 1.2 kb, λACL20은 0.7 kb, λACL21은

0.4 kb, λACL23은 0.9 kb, 그리고 λACL25는 3.04 kb와 0.6 kb (2개)의 cDNA가 삽입되어 있었고, λACL2, λACL6, λACL7, λACL10, λACL13, λACL14, λACL16, λACL18, λACL19, λACL22와 λACL24에서는 절단된 cDNA insert를 확인할 수 없었다. 이중에서 λACLB과 λACL12로부터 각각 1.0 kb와 3.0kb의 cDNA를 분리하여 plasmic인 pGEM-4Z vector에subcloning한 후, 이 plasmic를 probe로 사용하여 백서 간장세포의 핵 내에서 run-on전사 활성을 측정하였다. Insulin의 효과를 보기 위하여, 대조군, insulin 처치군 및 induction군의 백서 간장세포로부터 핵을 분리하여 소정의 방법(Ninial등, 1985)에 따라 [α-(32)**P]UTP를 사용하여 시험관 내에서 run-on전사를 시행한 후, 핵 내 총 RNA를 분리하고 pGEM-4Z vector, β-actin에 대한 cDNA가 삽입된 plasmic, pGACL8과 pGACL25-1을 각각 5μg씩 nitrocellulose 여과지에 slot blot한 후에 label된 총 RNA 일정량으로 hybridization 및 자가 방사법을 시행하였다. pGEM-4Z와 β-actin cDNA가 함유된 plasmid에서는 각군의 X-ray film상의 방사선 감광도에 있어서 변화가 없었으나, pGACLB과 PGACL25-1에서는insulin처치군은 대조군에 비하여 약 2배 증가하였으며, induction군에서는 오히려 감소현상을 보였다.

이상과 같은 결과로 미루어 보아 insulin처치 후 세포질 내 ACL mRNA증가는(Lee등, 1989) 1차적으로 insulin이 ACL gene의 전사 활성을 증가시키기 때문인 것으로 사료된다.

[영문]

This experiment was designed to illustrate the mechanism of regulation of ATP-citrate lyase (ACL)by insulin in rat liver. To determine whether the expression of ACL is regulated at pretranslational level, 120,000 λgtll-cDNA phages from rat liver cDNA library prepared by Kim et al (1989) were screened using purified anti-ACL antibody, and 25 positive clones containing the putative cDNA for ACL were identified and named λACL1 to λACL25, respectively. Phage DNAs were isolated from these 25 λACL clones and cDNA inserts were analyzed by agarose gel electrophoresis after digestion with EcoRI restriction enzyme. Fourteen out of 25 clones were identified as having cDNA inserts ranging from 0.4 to 3.0 kb in size. The sizs of cDNA inserts in λACL1, λACL3, and λACL4, λACL5, λACL8, λACL9, λACLll,

λACL17, λACL15, λACL17, λACL27, λACL71, λACL23, and λACL25 clone were 0.7 kb, 1.0 and 0.5 kb (2 inserts), 0.6 kb, 1.0 kb, 0.7 kb, 0.95 and 0.5 kb (2 inserts), 0.7 kb, 1.2 and 1.0 kb (2 inserts), 1.2 kb, 0.7 kb, 0.4 kb, 0.9 kb, and 3.0 and 0.6 kb (2 inserts), respectively, however the other 11 clones didn't show

the cDNA inserts in 1.2% agarose gel electrophoresis. Among the 14 positive clones, λACL8 and λACL25 which had cDNA inserts of 1.0 kb and 3.0 kb in size were further subcloned into EcoRI site af pGEM-4Z or pBR322 plasmids vector. After immobilization of these plasmids on nitrocellulose membrane, the amount of stop,

mRNA synthesized in isolated nuclei of rat liver by run-on transcription was measured, and it was found that the amount of mRNA synthesized in nuclei was increased 2 folds in the insulin-treated group after 3 hours of intraperitoneal insulin treatment, and decreased in the induction group after 48 hours of refeeding

with low fat and high carbohydrate diet, compared to the control group.

It was concluded that the increased mRNA level for ACL in rat liver cytosol after insulin treatment resulted from the increased rate of biosynthesis of myNA encoding the enzyme in nuclei.