Abstract

A complementary DNA (2510 bp) encoding a protein with high homology to mouse phospholipase A₂-activating protein (PLAP) was cloned from rat hepatocytes by differential screening of a subtractive (normoxic minus hypoxie) λGEM-2 cDNA library. This cDNA clone, denoted as HCW9, encodes a 647-amino-acid (aa) protein with 96.1% identity in the N-terminal portion (255 aa) to mouse PLAP [Clark et al., Proc. Natl. Acad. Sci. USA 88 (1991) 5418–5422]. Four cDNA clones were further isolated by screening a primary normoxic λGEM-2 cDNA library with a fragment of the HCW9 clone as probe. Partial sequencing of these clones revealed that all of the four clones were identical to clone HCW9. Northern blotting analysis of total RNA extracted from normoxic or hypoxic rat hepatocytes, with two different fragments of clone HCW9 as probes, demonstrated a single band with a mobility corresponding to a size of 2.5 kb, whose level was significantly decreased during chemical hypoxia.

Author Keywords: Hepatocyte; chemical hypoxia; bee venom melittin; recombinant DNA; phage library; PCR; nucleotide sequence; amino-acid sequence

Abbreviations: aa, amino acid(s); Ab, antibody(ies); bp, base pair(s); cDNA, DNA complementary to RNA; HCW9, protein (see Fig. 2) encoded by rat HCW9 clone (see section a); kb, kilobase(s) or 1000 bp; nt, nucleotide(s); ORF, open reading frame; PCR, polymerase chain reaction; pfu, plaque-forming unit(s); PL, phospholipase; PLAP, PLA₂-activating protein; SDS, sodium dodecyl sulfate; ss, single strand(ed); UTR, untranslated region(s)

Article Outline

• References