Verhoeven A J, Carling D, Jansen H
Department of Biochemistry, Erasmus University Rotterdam, The Netherlands.
J Lipid Res. 1994 Jun;35(6):966-75.
Rat adrenals contain a lipase activity that is indistinguishable from hepatic lipase (HL) present in liver. Expression of HL mRNA in adrenals was studied using the method of reverse transcription-polymerase chain reaction (RT-PCR). A 596-bp fragment of HL cDNA spanning exons 5 to 8 was amplified when using total RNA from rat adrenals and liver, but not from heart or kidney. The abundance of HL mRNA was quantified by competitive RT-PCR using a standard RNA that was generated in vitro by transcription from a deleted HL cDNA construct. Adrenals contained 0.4 attomoles of HL mRNA per microgram of total RNA, compared to 16 attomoles in liver. In hypertrophic adrenals isolated from corticotrophin-treated rats, the abundance also amounted to 0.4 attomoles of mRNA per microgram of total RNA. However, amplification of full-length cDNA from either control or hypertrophic adrenals was never observed. Detailed analysis by PCR using different combinations of primers indicated that exons 3 to 9 including the 3'-untranslated region were expressed in adrenal RNA, but not the first two coding exons. The upstream part of the adrenal lipase mRNA was cloned after rapid amplification of cDNA ends (RACE). The resulting clones showed a unique 126-bp sequence 5' of the exon 2-exon 3 junction. This sequence contained multiple termination codons in all three reading frames but lacked a potential start codon. RT-PCR using an HL-specific primer and an oligonucleotide directed against this 5'-sequence showed that it is not only expressed in RNA from adrenals but also in liver. Pulse-labeling of freshly isolated adrenocortical cells with [35S]methionine followed by immunoprecipitation with anti-HL antibodies failed to show synthesis of mature HL, but indicated the synthesis of immunoreactive proteins in the 40-45 kDa range that remained mainly intracellular. Hence, the HL gene is transcribed in adrenals but results in an mRNA species with a unique 5'-end. Translation from an internal start site may produce an intracellular HL isoform that differs markedly from the liver-type lipase previously identified in adrenals.
大鼠肾上腺含有一种脂肪酶活性，该活性与肝脏中存在的肝脂肪酶（HL）无法区分。使用逆转录-聚合酶链反应（RT-PCR）方法研究了HL mRNA在肾上腺中的表达。当使用来自大鼠肾上腺和肝脏的总RNA进行扩增时，可得到一个跨越外显子5至8的596 bp的HL cDNA片段，但心脏或肾脏的总RNA则无法扩增出该片段。使用通过从缺失的HL cDNA构建体体外转录产生的标准RNA，通过竞争性RT-PCR对HL mRNA的丰度进行定量。肾上腺每微克总RNA含有0.4阿托摩尔的HL mRNA，而肝脏中为16阿托摩尔。在从促肾上腺皮质激素处理的大鼠中分离出的肥大肾上腺中，每微克总RNA的mRNA丰度也为0.4阿托摩尔。然而，从未观察到从对照或肥大肾上腺中扩增出全长cDNA。使用不同引物组合进行PCR的详细分析表明，包括3'-非翻译区在内的外显子3至9在肾上腺RNA中表达，但前两个编码外显子不表达。通过cDNA末端快速扩增（RACE）克隆了肾上腺脂肪酶mRNA的上游部分。所得克隆在第2外显子与第3外显子连接处的5'端显示出一个独特的126 bp序列。该序列在所有三个阅读框中均包含多个终止密码子，但缺乏潜在的起始密码子。使用HL特异性引物和针对该5'-序列的寡核苷酸进行RT-PCR表明，它不仅在肾上腺RNA中表达，也在肝脏中表达。用[35S]甲硫氨酸对新鲜分离的肾上腺皮质细胞进行脉冲标记，然后用抗HL抗体进行免疫沉淀，未显示出成熟HL的合成，但表明在40-45 kDa范围内合成了主要保留在细胞内的免疫反应性蛋白。因此，HL基因在肾上腺中被转录，但产生具有独特5'-末端的mRNA种类。从内部起始位点进行翻译可能会产生一种细胞内HL同工型，该同工型与先前在肾上腺中鉴定出的肝型脂肪酶明显不同。
