Differential screening of a cDNA library with cDNA probes amplified in a heterologous host: isolation of murine GRP78 (BiP) and other serum-regulated low-abundance mRNAs.
A novel method was used to screen differentially a cDNA library for clones representing serum-regulated mRNA species of low abundance. To increase the amount of probe available for screening, the cDNA probe was cloned and amplified. Two separate cDNA 'probe' libraries were constructed in the Escherichia coli plasmid vector pDE613, using poly(A)+mRNA from murine cells at 0 and 16 h after stimulation of a G0 population. Radiolabelled plasmid DNA from each library was hybridized sequentially to colony blots of the third 'target' library, constructed with mRNA from serum-stimulated cells in the Bacillus subtilis vector pBD214. Differential screening of the target cDNA library with the two probe libraries identified novel murine cDNA clones, some representing cytoplasmic poly(A)+RNA species of low (0.01%) abundance, accumulating after serum stimulation of a quiescent mouse embryo fibroblast population. One cDNA clone was found to correspond to mitochondrial 16S rRNA and a second was identified as the murine equivalent of previously described cDNA clones for the hamster 78-kDa glucose-regulated protein (GRP78) and the rat immunoglobulin heavy-chain-binding protein. GRP78 mRNA has not previously been recognized as a serum-inducible message.
Parfett
C L
CL
Cancer Research Laboratory, University of Western Ontario, London, Canada.
Hofbauer
R
R
Brudzynski
K
K
Edwards
D R
DR
Denhardt
D T
DT
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics*; D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods*; D006224:Cricetinae; D004247:DNA / Q000235:genetics*; D015342:DNA Probes; D000091342:Endoplasmic Reticulum Chaperone BiP; D005784:Gene Amplification; D015723:Gene Library; D006360:Heat-Shock Proteins; D051379:Mice; D008807:Mice, Inbred BALB C; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics*; D051381:Rats; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity
D002352:Carrier Proteins; D015342:DNA Probes; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; C000717248:Hspa5 protein, mouse; D018832:Molecular Chaperones; D012333:RNA, Messenger; D004247:DNA