Detection of Proopiomelanocortin MRNA by in Situ Hybridization with an Oligonucleotide Probe

Overview

Journal Proc Natl Acad Sci U S A

Specialty Science

Date 1986 Aug 1

PMID 3461440

Citations 21

Authors

M E Lewis

T G Sherman

S Burke

H Akil

L G Davis

R Arentzen

S J Watson

Affiliations

Soon will be listed here.

Abstract

Synthetic oligonucleotide probes can be easily obtained and used, in contrast to cDNA cloning to develop probes, and thus the present study was carried out to determine whether such probes could also be useful for in situ hybridization. A 24-base synthetic oligonucleotide complementary to part of the alpha-melanocyte-stimulating hormone (alpha-MSH) coding region of proopiomelanocortin (POMC) mRNA was 5'-end-labeled by using [gamma-32P]ATP with T4 polynucleotide kinase or was 3' tailed by using [alpha-32P]dATP or [3H]dCTP with terminal deoxynucleotidyltransferase. Blot analysis of pituitary poly(A)+ RNA showed that the oligonucleotide hybridized to a single species with a molecular size of approximately 1200 nucleotides, consistent with that determined previously for POMC mRNA. The oligonucleotide, regardless of labeling method, hybridized to cells in the pituitary intermediate lobe, but not in the posterior lobe. Only the 3H-labeled probe gave resolution of individual pituitary anterior lobe cells. The specificity of the hybridization was determined by showing that the intermediate lobe signal was blocked by prehybridization of the tissue with unlabeled alpha-MSH oligonucleotide probe. Furthermore, the hybridized probe exhibited a sharp sigmoid curve when melted off. Finally, the oligonucleotide probe detected, in situ, the haloperidol-induced elevation of intermediate lobe POMC mRNA. Thus, the oligonucleotide probe exhibited hybridization in an anatomically and biochemically specific manner, and it detected a tissue-specific change in mRNA levels in situ.

Citing Articles

Cellular Profiles of Prodynorphin and Preproenkephalin mRNA-Expressing Neurons in the Anterior Olfactory Tubercle of Mice.

Maegawa A, Murata K, Kuroda K, Fujieda S, Fukazawa Y Front Neural Circuits. 2022; 16:908964.

PMID: 35937204 PMC: 9352893. DOI: 10.3389/fncir.2022.908964.

Ribosomal RNA Modulates Aggregation of the Prion Protein HET-s.

Pang Y, Kovachev P, Sanyal S Int J Mol Sci. 2020; 21(17).

PMID: 32882892 PMC: 7504336. DOI: 10.3390/ijms21176340.

The antiprion compound 6-aminophenanthridine inhibits the protein folding activity of the ribosome by direct competition.

Pang Y, Kurella S, Voisset C, Samanta D, Banerjee D, Schabe A J Biol Chem. 2013; 288(26):19081-9.

PMID: 23673663 PMC: 3696681. DOI: 10.1074/jbc.M113.466748.

Growth hormone (GH) and prolactin (PRL) gene expression and immunoreactivity in GH- and PRL-producing human pituitary adenomas.

Li J, Stefaneanu L, Kovacs K, Horvath E, Smyth H Virchows Arch A Pathol Anat Histopathol. 1993; 422(3):193-201.

PMID: 8493775 DOI: 10.1007/BF01621802.

c-fos oncogene underexpression in salivary gland tumors as measured by in situ hybridization.

Birek C, Lui E, Dardick I Am J Pathol. 1993; 142(3):917-23.

PMID: 8456948 PMC: 1886811.

References

Uhl G, Zingg H, Habener J . Vasopressin mRNA in situ hybridization: localization and regulation studied with oligonucleotide cDNA probes in normal and Brattleboro rat hypothalamus. Proc Natl Acad Sci U S A. 1985; 82(16):5555-9. PMC: 391161. DOI: 10.1073/pnas.82.16.5555. View

Pelletier G, LECLERC R, Labrie F, Cote J, Chretien M, Lis M . Immunohistochemical localization of beta-lipotropic hormone in the pituitary gland. Endocrinology. 1977; 100(3):770-6. DOI: 10.1210/endo-100-3-770. View

Lewis M, Sherman T, Watson S . In situ hybridization histochemistry with synthetic oligonucleotides: strategies and methods. Peptides. 1985; 6 Suppl 2:75-87. DOI: 10.1016/0196-9781(85)90138-x. View

Kelsey J, Watson S, Burke S, Akil H, Roberts J . Characterization of proopiomelanocortin mRNA detected by in situ hybridization. J Neurosci. 1986; 6(1):38-42. PMC: 6568624. View

Davis L, Arentzen R, Reid J, Manning R, Wolfson B, Lawrence K . Glucocorticoid sensitivity of vasopressin mRNA levels in the paraventricular nucleus of the rat. Proc Natl Acad Sci U S A. 1986; 83(4):1145-9. PMC: 323028. DOI: 10.1073/pnas.83.4.1145. View

Sherman T, McKelvy J, Watson S . Vasopressin mRNA regulation in individual hypothalamic nuclei: a northern and in situ hybridization analysis. J Neurosci. 1986; 6(6):1685-94. PMC: 6568715. View

Harrison P, CONKIE D, Paul J, Jones K . Localisation of cellular globin messenger RNA by in situ hybridisation to complementary DNA. FEBS Lett. 1973; 32(1):109-12. DOI: 10.1016/0014-5793(73)80749-5. View

Bonner T, Brenner D, Neufeld B, Britten R . Reduction in the rate of DNA reassociation by sequence divergence. J Mol Biol. 1973; 81(2):123-35. DOI: 10.1016/0022-2836(73)90184-8. View

Britten R, Graham D, Neufeld B . Analysis of repeating DNA sequences by reassociation. Methods Enzymol. 1974; 29:363-418. DOI: 10.1016/0076-6879(74)29033-5. View

10.

BLOOM F, Battenberg E, Rossier J, Ling N, Leppaluoto J, Vargo T . Endorphins are located in the intermediate and anterior lobes of the pituitary gland, not in the neurohypophysis. Life Sci. 1977; 20(1):43-7. DOI: 10.1016/0024-3205(77)90126-6. View

11.

Fritz H, Belagaje R, Brown E, Fritz R, Jones R, Lees R . High-pressure liquid chromatography in polynucleotide synthesis. Biochemistry. 1978; 17(7):1257-67. DOI: 10.1021/bi00600a020. View

12.

Brahic M, Haase A . Detection of viral sequences of low reiteration frequency by in situ hybridization. Proc Natl Acad Sci U S A. 1978; 75(12):6125-9. PMC: 393131. DOI: 10.1073/pnas.75.12.6125. View

13.

Nakanishi S, Inoue A, Kita T, Nakamura M, Chang A, Cohen S . Nucleotide sequence of cloned cDNA for bovine corticotropin-beta-lipotropin precursor. Nature. 1979; 278(5703):423-7. DOI: 10.1038/278423a0. View

14.

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

15.

McWilliams D, Boime I . Cytological localization of placental lactogen messenger ribonucleic acid in syncytiotrophoblast layers of human placenta. Endocrinology. 1980; 107(3):761-5. DOI: 10.1210/endo-107-3-761. View

16.

Thomas P . Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980; 77(9):5201-5. PMC: 350025. DOI: 10.1073/pnas.77.9.5201. View

17.

Hudson P, Penschow J, Shine J, Ryan G, NIALL H, COGHLAN J . Hybridization histochemistry: use of recombinant DNA as a "homing probe" for tissue localization of specific mRNA populations. Endocrinology. 1981; 108(1):353-6. DOI: 10.1210/endo-108-1-353. View

18.

Pochet R, Brocas H, Vassart G, Toubeau G, Seo H, Refetoff S . Radioautographic localization of prolactin messenger RNA on histological sections by in situ hybridization. Brain Res. 1981; 211(2):433-8. DOI: 10.1016/0006-8993(81)90969-0. View

19.

Angerer L, Angerer R . Detection of poly A+ RNA in sea urchin eggs and embryos by quantitative in situ hybridization. Nucleic Acids Res. 1981; 9(12):2819-40. PMC: 326896. DOI: 10.1093/nar/9.12.2819. View

20.

Schachter B, Johnson L, Baxter J, Roberts J . Differential regulation by glucocorticoids of proopiomelanocortin mRNA levels in the anterior and intermediate lobes of the rat pituitary. Endocrinology. 1982; 110(4):1442-4. DOI: 10.1210/endo-110-4-1442. View