ALTERATION IN CHROMATIN CONFORMATION OF PROLIFERATING AND DIFFERENTIATING ENZYME GENES IN NORMAL MOUSE LIVER AND ASCITES HEPATOMA CELLS AND ITS RELATION TO THE GENE EXPRESSION OF ENZYMES

Nuclei from the normal mouse liver were partially digested with micrococcal nuclease, followed by DNA extraction, agarose gel clectrophoresis and dot blot hybridization with ~(32)P-labeled cDNA probes of CPS_1 and ACT complex, It was clearly shown that the CPS_1 genes were distributed on the monomer, dimer. and trimer of nucleosomes, while the genes coding for ACT complex were distributed on the condensed oligonucleosomes. An opposite manner of distribution of CPS_1 and ACT complex genes was, however, noted in the case of ascites hepatoma cells, in which the specific activity of ACT was 13 times higher than that in the normal liver, while that of CPS_1 was remarkably reduced. Similar patterns of change in mRNA level of CPS_1 and ACT complex were observed in the normal mouse liver and ascites hepatoma cells, indicating a close relationship between chromatin structure and gene expression of these enzymes.     

STUDIES ON THE MECHANISM OF CARCINOGENESIS(IX)——DIFFERENTIAL EXPRESSION OF PROLIFERATING AND TISSUE-SPECIFIC ENZYME GENES-DURING HEPATOCARCINOGENESIS

With cDNA fragments of CPSI, OCT and ACT as probes, dot and Northern blot analyses of poly(A)+-RNA from rat liver with different pathological lesions during carcinogenesis induced by di-ethylnitrosamine were conducted. It was shown that the level of mRNA of tissue-specific enzymes, CPSI and OCT decreased while that of the proliferating enzyme ACT mRNA increased, and the alteration was correlated with the degree of pathological changes in each case. The relative changes in the mRNA level of these enzymes during hepatocarcinogenesis were correlated with that of enzyme activities. Implication of these findings in the mechanism of carcinogenesis in terms of cell proliferation and differentiation was discussed.     

磷酰胺類化合物的製備

本文提出一制备磷酰胺类化合物的新法。将一克分子二氯磷酰胺类化合物与二克分子甲酸在乾燥有机溶剂中加热回流。此法的特点在於产量高,产物容易分离。利用此法,目前已制得苯胺,对-氯苯胺,α-萘胺,对-磺酰氨基苯胺,隣-甲氧基苯胺,对-甲基苯胺,以及苯甲胺的磷酰胺化合物:但不能制得对-硝基苯胺及对-氨基苯甲酸的磷酰胺化合物。对-氨基苯甲酸的磷酰胺化合物可由N-对-氯甲酰苯基二氯磷酰胺与甲酸反应制得,对-硝基苯胺的磷酰胺则始终没有制备成功。     

小鼠腹水肝癌细胞增殖酶及分化酶基因染色质构象的变化及其与酶基因表达的关系

小鼠腹水肝癌细胞的CPS_1活性较正常肝显著降低,而ACT活性则增高,约为正常肝的13倍。RNA-cDNA斑点杂交证明,肝癌中CPS_1mRNA很低,而ACTmRNA较正常肝增多。以微球菌核酸酶限制性消化正常肝及肝癌细胞核,琼脂糖凝胶电泳以及DNA-cDNA斑点杂交结果表明,在正常肝中,表达活跃的CPS_1基因分布在结构松散的单体、二聚体、三聚体核小体上,而表达较低的ACT基因则分布在比较致密的寡聚核小体上。腹水肝癌细胞的情况正好相反。CPS_1基因的表达很低,主要分布于寡聚核小体,而ACT基因的表达增高,主要分布于单体核小体。这些结果充分说明,肝癌细胞中分化酶CPS_1基因与增殖酶AST基因表达的相互变化与这两种基因核小体构象的相互变化有密切关系。     

STUDIES ON DNA SEQUENCE SPECIFIC BINDING PROTEINS FOR 5'''' FLANKING UPSTREAM REGION OF CPSI GENE

Two specific carbamyl phosphate synthetase Ⅰ gene binding nuclear proteins (M. W. 109kD and 74 kD) have been determined in the rat liver by the protein blotting technique (South-western blot assay). The result shows that they are not present in the normal rat spleenand F-26 rat hepatoma cell. The Ba131 nuclease deletion in the CPSI gene 5' upstream regionproves that the binding sites for 109 kD and 74 kD are respectively located in the regions of- 38 bp to - 4 bp and - 113 bp to -38 bp. The binding proteins may be the liver-specific onesof the CPSI gene, which are related to hepatocyte differentiation and hepatocarcinogenesis.     

氨甲酰磷酸合成酶Ⅰ基因5′端上游DNA特异结合蛋白的研究

本文利用Southwestern blot法检测了CPSI基因5′端上游序列特异结合蛋白,结果发现,大鼠肝细胞核蛋白中存在109kD和74kD的DNA结合蛋白,Bal 31酶切序列缺失实验证明,109kD和74kD蛋白质的结合位点分别位于—38至—4bp及—113至—38bp区域,109kD蛋白质还与—480至—161bp区结合,但在大鼠脾及F26大鼠肝癌细胞中未被测出.109kD和74kD DNA结合蛋白可能是肝组织特异的CPSI基因结合蛋白,与肝细胞的分化及癌变有关。     

CLONING OF cDNA CODING FOR CARBAMYL PHOSPHATE SYNTHETASE I AND CHANGES IN LEVELS OF CPS_1 mRNA DURING HEPATOCARCINOGENESIS

cDNA coding for carbamyl phosphate synthetase I was cloned from recombinant plasmid with insertcomplementary to the mRNA for CPS_1 followed by hybrid-selected translation screening. The length ofthe insert CPS_1 cDNA was approximately 800 base pairs. Using this cDNA as a probe, it was foundby dot-blot analysis of the total RNAs and poly(A)~+-RNAs isolated from rat livers with different path-ological lesions induced by diethylnitrosamine that the levels of CPS_1 mRNA were decreased, the de-crease being correlated with the malignancy of hepatocytes during carcinogenesis.     

CPS_1 cDNA克隆及大鼠肝癌癌变过程中CPS_1 mRNA量的变化

本研究以CPS_1mRNA为模板合成cDNA,与质粒pBR 322 DNA重组,经选择杂交法筛选获得GPS_1 cDNA克隆。以~(32)P标记cDNA作为探针与二乙基亚硝胺(DENA)诱发大鼠肝癌过程中不同病变肝中提取的总RNA及poly(A)~+RNA杂交。结果发现,CPS_1 mRNA的量随肝细胞癌变程度加深而递减,说明肝癌癌变过程中,CPS_1 基因表达的异常可能发生在转录水平。     

癌变原理研究——Ⅸ.大鼠肝癌癌变过程中细胞增殖酶和组织特异酶基因表达的相互变化

以CPS_1,OCT,ACT cDNA克隆片段为探针与二乙基亚硝胺诱发大鼠肝癌癌变过程不同病变肝组织的poly(A)~+-RNA进行斑点杂交及Northern印迹杂交。结果表明:组织特异酶CPS_1,OCT mRNA量降低,而细胞增殖酶ACT mRNA量升高。这些mRNA量的变化都随病变程度而加深。CPS_1 mRNA分子略大于28S,OCT mRNA约为15S,ACT mRNA分子约为35S。癌变过程中CPS_1,OCT以及ACT mRNA量的相互变化与不同癌变肝组织酶活性的相互变化呈相关关系。