Hepatic lipase gene is transcribed in rat adrenals into a truncated mRNA.
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.
Verhoeven
A J
AJ
Department of Biochemistry, Erasmus University Rotterdam, The Netherlands.
Carling
D
D
Jansen
H
H
D000302:Adrenal Cortex / Q000378:metabolism; D000311:Adrenal Glands / Q000378:metabolism* / Q000473:pathology; D000818:Animals; D001483:Base Sequence; D005091:Exons; D006984:Hypertrophy; D008049:Lipase / Q000235:genetics*; D008099:Liver / Q000201:enzymology*; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase; D051381:Rats; D017208:Rats, Wistar; D014158:Transcription, Genetic
D012333:RNA, Messenger; D012194:RNA-Directed DNA Polymerase; D008049:Lipase