Phenol emulsion-enhanced DNA-driven subtractive cDNA cloning: isolation of low-abundance monkey cortex-specific mRNAs.

AUTOR(ES)

Travis, G H

RESUMO

To isolate cDNA clones of low-abundance mRNAs expressed in monkey cerebral cortex but absent from cerebellum, we developed an improved subtractive cDNA cloning procedure that requires only modest quantities of mRNA. Plasmid DNA from a monkey cerebellum cDNA library was hybridized in large excess to radiolabeled monkey cortex cDNA in a phenol emulsion-enhanced reaction. The unhybridized cortex cDNA was isolated by chromatography on hydroxyapatite and used to probe colonies from a monkey cortex cDNA library. Of 60,000 colonies screened, 163 clones were isolated and confirmed by colony hybridization or RNA blotting to represent mRNAs, ranging from 0.001% to 0.1% abundance, specific to or highly enriched in cerebral cortex relative to cerebellum. Clones of one medium-abundance mRNA were recovered almost quantitatively. Two of the lower-abundance mRNAs were expressed at levels reduced by a factor of 10 in Alzheimer disease relative to normal human cortex. One of these was identified as the monkey preprosomatostatin I mRNA.

Documentos Relacionados

• Purification of low-abundance messenger RNAs from rat liver by polysome immunoadsorption.
• Enhanced Function Conferred on Low-Abundance Chemoreceptor Trg by a Methyltransferase-Docking Site
• Molecular cloning of developmentally regulated, low-abundance mRNA sequences from embryonic muscle.
• Molecular cloning of partial cDNA copies of two distinct mouse IFN-beta mRNAs.
• cDNA cloning and nucleotide sequence comparison of Chinese hamster metallothionein I and II mRNAs.