Phenol Emulsion-enhanced DNA-driven Subtractive CDNA Cloning: Isolation of Low-abundance Monkey Cortex-specific MRNAs

Overview

Journal Proc Natl Acad Sci U S A

Specialty Science

Date 1988 Mar 1

PMID 2894033

Citations 20

Authors

G H Travis

J G Sutcliffe

Affiliations

Soon will be listed here.

Abstract

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.

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References

Kohne D, LEVISON S, Byers M . Room temperature method for increasing the rate of DNA reassociation by many thousandfold: the phenol emulsion reassociation technique. Biochemistry. 1977; 16(24):5329-41. DOI: 10.1021/bi00643a026. View

Chirgwin J, Przybyla A, MacDonald R, Rutter W . Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979; 18(24):5294-9. DOI: 10.1021/bi00591a005. View

Gergen J, STERN R, Wensink P . Filter replicas and permanent collections of recombinant DNA plasmids. Nucleic Acids Res. 1979; 7(8):2115-36. PMC: 342374. DOI: 10.1093/nar/7.8.2115. View

Grunstein M, Wallis J . Colony hybridization. Methods Enzymol. 1979; 68:379-89. DOI: 10.1016/0076-6879(79)68027-8. View

Dworkin M, Dawid I . Use of a cloned library for the study of abundant poly(A)+RNA during Xenopus laevis development. Dev Biol. 1980; 76(2):449-64. DOI: 10.1016/0012-1606(80)90393-0. View

Timberlake W . Developmental gene regulation in Aspergillus nidulans. Dev Biol. 1980; 78(2):497-510. DOI: 10.1016/0012-1606(80)90349-8. View

Morrison J, Rogers J, Scherr S, Benoit R, Bloom F . Somatostatin immunoreactivity in neuritic plaques of Alzheimer's patients. Nature. 1985; 314(6006):90-2. DOI: 10.1038/314090a0. View

Roberts G, Crow T, Polak J . Location of neuronal tangles in somatostatin neurones in Alzheimer's disease. Nature. 1985; 314(6006):92-4. DOI: 10.1038/314092a0. View

Milner R, Lai C, Nave K, Lenoir D, Ogata J, Sutcliffe J . Nucleotide sequences of two mRNAs for rat brain myelin proteolipid protein. Cell. 1985; 42(3):931-9. DOI: 10.1016/0092-8674(85)90289-2. View

10.

Tsou A, Lai C, Danielson P, Noonan D, Sutcliffe J . Structural characterization of a heterogeneous family of rat brain mRNAs. Mol Cell Biol. 1986; 6(3):768-78. PMC: 367577. DOI: 10.1128/mcb.6.3.768-778.1986. View

11.

Travis G, Naus C, Morrison J, Bloom F, Sutcliffe J . Subtractive cloning of complementary DNAs and analysis of messenger RNAs with regional heterogeneous distributions in primate cortex. Neuropharmacology. 1987; 26(7B):845-54. DOI: 10.1016/0028-3908(87)90061-x. View

12.

Aviv H, Leder P . Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc Natl Acad Sci U S A. 1972; 69(6):1408-12. PMC: 426713. DOI: 10.1073/pnas.69.6.1408. View

13.

Schibler U, Tosi M, Pittet A, Fabiani L, Wellauer P . Tissue-specific expression of mouse alpha-amylase genes. J Mol Biol. 1980; 142(1):93-116. DOI: 10.1016/0022-2836(80)90208-9. View

14.

Davies P, Katzman R, Terry R . Reduced somatostatin-like immunoreactivity in cerebral cortex from cases of Alzheimer disease and Alzheimer senile dementa. Nature. 1980; 288(5788):279-80. DOI: 10.1038/288279a0. View

15.

Shen L, Pictet R, Rutter W . Human somatostatin I: sequence of the cDNA. Proc Natl Acad Sci U S A. 1982; 79(15):4575-9. PMC: 346717. DOI: 10.1073/pnas.79.15.4575. View

16.

Okayama H, Berg P . A cDNA cloning vector that permits expression of cDNA inserts in mammalian cells. Mol Cell Biol. 1983; 3(2):280-9. PMC: 368532. DOI: 10.1128/mcb.3.2.280-289.1983. View

17.

Feinberg A, Vogelstein B . A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983; 132(1):6-13. DOI: 10.1016/0003-2697(83)90418-9. View