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. 1994 Dec 20;91(26):12696–12700. doi: 10.1073/pnas.91.26.12696

Allele-specific quantification of Drosophila engrailed and invected transcripts.

A S Goldsborough 1, T B Kornberg 1
PMCID: PMC45506  PMID: 7809104

Abstract

Changes in levels of transcription can be difficult to gauge in animals with lethal mutations. For example, mutations in a regulatory region of an essential gene can have secondary consequences that complicate attempts to quantify the transcripts produced by the mutant gene. We describe a method that circumvents this problem by revealing the relative amount of transcript produced from each allele in a heterozygote. With this method, recessive lethal mutations can be analyzed in animals that are phenotypically wild type. We used this technique to analyze mutations in the regulatory region of the Drosophila engrailed gene and found that truncations reduce transcription to levels that depend both upon the tissue and upon the location of the chromosomal break. We also found that these mutations affect expression of the linked invected gene, suggesting that engrailed and invected share a complex set of regulatory elements that operate over at least 85 kb. We suggest that this technique will have general utility for the quantitation of allele-specific transcripts, even when amounts of tissue are limiting.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Barker G. F., Beemon K. Rous sarcoma virus RNA stability requires an open reading frame in the gag gene and sequences downstream of the gag-pol junction. Mol Cell Biol. 1994 Mar;14(3):1986–1996. doi: 10.1128/mcb.14.3.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brook J. D., McCurrach M. E., Harley H. G., Buckler A. J., Church D., Aburatani H., Hunter K., Stanton V. P., Thirion J. P., Hudson T. Molecular basis of myotonic dystrophy: expansion of a trinucleotide (CTG) repeat at the 3' end of a transcript encoding a protein kinase family member. Cell. 1992 Feb 21;68(4):799–808. doi: 10.1016/0092-8674(92)90154-5. [DOI] [PubMed] [Google Scholar]
  3. Chess A., Simon I., Cedar H., Axel R. Allelic inactivation regulates olfactory receptor gene expression. Cell. 1994 Sep 9;78(5):823–834. doi: 10.1016/s0092-8674(94)90562-2. [DOI] [PubMed] [Google Scholar]
  4. Coleman K. G., Poole S. J., Weir M. P., Soeller W. C., Kornberg T. The invected gene of Drosophila: sequence analysis and expression studies reveal a close kinship to the engrailed gene. Genes Dev. 1987 Mar;1(1):19–28. doi: 10.1101/gad.1.1.19. [DOI] [PubMed] [Google Scholar]
  5. Drees B., Ali Z., Soeller W. C., Coleman K. G., Poole S. J., Kornberg T. The transcription unit of the Drosophila engrailed locus: an unusually small portion of a 70,000 bp gene. EMBO J. 1987 Sep;6(9):2803–2809. doi: 10.1002/j.1460-2075.1987.tb02576.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Foley K. P., Leonard M. W., Engel J. D. Quantitation of RNA using the polymerase chain reaction. Trends Genet. 1993 Nov;9(11):380–385. doi: 10.1016/0168-9525(93)90137-7. [DOI] [PubMed] [Google Scholar]
  7. Fu Y. H., Friedman D. L., Richards S., Pearlman J. A., Gibbs R. A., Pizzuti A., Ashizawa T., Perryman M. B., Scarlato G., Fenwick R. G., Jr Decreased expression of myotonin-protein kinase messenger RNA and protein in adult form of myotonic dystrophy. Science. 1993 Apr 9;260(5105):235–238. doi: 10.1126/science.8469976. [DOI] [PubMed] [Google Scholar]
  8. Fu Y. H., Pizzuti A., Fenwick R. G., Jr, King J., Rajnarayan S., Dunne P. W., Dubel J., Nasser G. A., Ashizawa T., de Jong P. An unstable triplet repeat in a gene related to myotonic muscular dystrophy. Science. 1992 Mar 6;255(5049):1256–1258. doi: 10.1126/science.1546326. [DOI] [PubMed] [Google Scholar]
  9. Gilliland G., Perrin S., Blanchard K., Bunn H. F. Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2725–2729. doi: 10.1073/pnas.87.7.2725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hama C., Ali Z., Kornberg T. B. Region-specific recombination and expression are directed by portions of the Drosophila engrailed promoter. Genes Dev. 1990 Jul;4(7):1079–1093. doi: 10.1101/gad.4.7.1079. [DOI] [PubMed] [Google Scholar]
  11. Holliday R. Genomic imprinting and allelic exclusion. Dev Suppl. 1990:125–129. [PubMed] [Google Scholar]
  12. Jaynes J. B., O'Farrell P. H. Active repression of transcription by the engrailed homeodomain protein. EMBO J. 1991 Jun;10(6):1427–1433. doi: 10.1002/j.1460-2075.1991.tb07663.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kuner J. M., Nakanishi M., Ali Z., Drees B., Gustavson E., Theis J., Kauvar L., Kornberg T., O'Farrell P. H. Molecular cloning of engrailed: a gene involved in the development of pattern in Drosophila melanogaster. Cell. 1985 Aug;42(1):309–316. doi: 10.1016/s0092-8674(85)80126-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lim S. K., Sigmund C. D., Gross K. W., Maquat L. E. Nonsense codons in human beta-globin mRNA result in the production of mRNA degradation products. Mol Cell Biol. 1992 Mar;12(3):1149–1161. doi: 10.1128/mcb.12.3.1149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Mahadevan M., Tsilfidis C., Sabourin L., Shutler G., Amemiya C., Jansen G., Neville C., Narang M., Barceló J., O'Hoy K. Myotonic dystrophy mutation: an unstable CTG repeat in the 3' untranslated region of the gene. Science. 1992 Mar 6;255(5049):1253–1255. doi: 10.1126/science.1546325. [DOI] [PubMed] [Google Scholar]
  16. Nelson D. L., Warren S. T. Trinucleotide repeat instability: when and where? Nat Genet. 1993 Jun;4(2):107–108. doi: 10.1038/ng0693-107. [DOI] [PubMed] [Google Scholar]
  17. Robey E., Fowlkes B. J. Selective events in T cell development. Annu Rev Immunol. 1994;12:675–705. doi: 10.1146/annurev.iy.12.040194.003331. [DOI] [PubMed] [Google Scholar]
  18. Roses A. D. Muscle biochemistry and a genetic study of myotonic dystrophy. Science. 1994 Apr 22;264(5158):587–588. doi: 10.1126/science.8160018. [DOI] [PubMed] [Google Scholar]
  19. Roses A. D. Muscle biochemistry and a genetic study of myotonic dystrophy. Science. 1994 Apr 22;264(5158):587–588. doi: 10.1126/science.8160018. [DOI] [PubMed] [Google Scholar]
  20. Sabouri L. A., Mahadevan M. S., Narang M., Lee D. S., Surh L. C., Korneluk R. G. Effect of the myotonic dystrophy (DM) mutation on mRNA levels of the DM gene. Nat Genet. 1993 Jul;4(3):233–238. doi: 10.1038/ng0793-233. [DOI] [PubMed] [Google Scholar]
  21. Schiavi S. C., Wellington C. L., Shyu A. B., Chen C. Y., Greenberg M. E., Belasco J. G. Multiple elements in the c-fos protein-coding region facilitate mRNA deadenylation and decay by a mechanism coupled to translation. J Biol Chem. 1994 Feb 4;269(5):3441–3448. [PubMed] [Google Scholar]
  22. Weksberg R., Shen D. R., Fei Y. L., Song Q. L., Squire J. Disruption of insulin-like growth factor 2 imprinting in Beckwith-Wiedemann syndrome. Nat Genet. 1993 Oct;5(2):143–150. doi: 10.1038/ng1093-143. [DOI] [PubMed] [Google Scholar]

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