Oligonucleotide probes detect splicing variants in situ in Drosophila embryos (original) (raw)

Abstract

We describe a method for the in situ detection of specific splicing variants. The method is based on the use of antisense oligonucleotides designed to span splice junctions labelled with digoxigenin by terminal transferase tailing. We find that the spatial patterns of Ubx splicing variants Ia and IIa are similar in early embryos, but differ in late embryos. Variant IVa is only detected in the CNS (ps6) at stages 16 and 17. We also present evidence indicating that the first splicing event is cotranscriptional.

5687

Images in this article

Selected References

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

  1. Akam M. E., Martinez-Arias A. The distribution of Ultrabithorax transcripts in Drosophila embryos. EMBO J. 1985 Jul;4(7):1689–1700. doi: 10.1002/j.1460-2075.1985.tb03838.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Akam M. E. The location of Ultrabithorax transcripts in Drosophila tissue sections. EMBO J. 1983;2(11):2075–2084. doi: 10.1002/j.1460-2075.1983.tb01703.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bermingham J. R., Jr, Martinez-Arias A., Petitt M. G., Scott M. P. Different patterns of transcription from the two Antennapedia promoters during Drosophila embryogenesis. Development. 1990 Jul;109(3):553–566. doi: 10.1242/dev.109.3.553. [DOI] [PubMed] [Google Scholar]
  4. Bermingham J. R., Jr, Scott M. P. Developmentally regulated alternative splicing of transcripts from the Drosophila homeotic gene Antennapedia can produce four different proteins. EMBO J. 1988 Oct;7(10):3211–3222. doi: 10.1002/j.1460-2075.1988.tb03188.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Breitbart R. E., Andreadis A., Nadal-Ginard B. Alternative splicing: a ubiquitous mechanism for the generation of multiple protein isoforms from single genes. Annu Rev Biochem. 1987;56:467–495. doi: 10.1146/annurev.bi.56.070187.002343. [DOI] [PubMed] [Google Scholar]
  6. Cribbs D. L., Pultz M. A., Johnson D., Mazzulla M., Kaufman T. C. Structural complexity and evolutionary conservation of the Drosophila homeotic gene proboscipedia. EMBO J. 1992 Apr;11(4):1437–1449. doi: 10.1002/j.1460-2075.1992.tb05188.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gavis E. R., Hogness D. S. Phosphorylation, expression and function of the Ultrabithorax protein family in Drosophila melanogaster. Development. 1991 Aug;112(4):1077–1093. doi: 10.1242/dev.112.4.1077. [DOI] [PubMed] [Google Scholar]
  8. Hafen E., Levine M., Garber R. L., Gehring W. J. An improved in situ hybridization method for the detection of cellular RNAs in Drosophila tissue sections and its application for localizing transcripts of the homeotic Antennapedia gene complex. EMBO J. 1983;2(4):617–623. doi: 10.1002/j.1460-2075.1983.tb01472.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kornfeld K., Saint R. B., Beachy P. A., Harte P. J., Peattie D. A., Hogness D. S. Structure and expression of a family of Ultrabithorax mRNAs generated by alternative splicing and polyadenylation in Drosophila. Genes Dev. 1989 Feb;3(2):243–258. doi: 10.1101/gad.3.2.243. [DOI] [PubMed] [Google Scholar]
  10. Lopez A. J., Hogness D. S. Immunochemical dissection of the Ultrabithorax homeoprotein family in Drosophila melanogaster. Proc Natl Acad Sci U S A. 1991 Nov 15;88(22):9924–9928. doi: 10.1073/pnas.88.22.9924. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Mann R. S., Hogness D. S. Functional dissection of Ultrabithorax proteins in D. melanogaster. Cell. 1990 Feb 23;60(4):597–610. doi: 10.1016/0092-8674(90)90663-y. [DOI] [PubMed] [Google Scholar]
  12. Mlodzik M., Fjose A., Gehring W. J. Molecular structure and spatial expression of a homeobox gene from the labial region of the Antennapedia-complex. EMBO J. 1988 Aug;7(8):2569–2578. doi: 10.1002/j.1460-2075.1988.tb03106.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. O'Connor M. B., Binari R., Perkins L. A., Bender W. Alternative RNA products from the Ultrabithorax domain of the bithorax complex. EMBO J. 1988 Feb;7(2):435–445. doi: 10.1002/j.1460-2075.1988.tb02831.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Schmitz G. G., Walter T., Seibl R., Kessler C. Nonradioactive labeling of oligonucleotides in vitro with the hapten digoxigenin by tailing with terminal transferase. Anal Biochem. 1991 Jan;192(1):222–231. doi: 10.1016/0003-2697(91)90212-c. [DOI] [PubMed] [Google Scholar]
  15. Shermoen A. W., O'Farrell P. H. Progression of the cell cycle through mitosis leads to abortion of nascent transcripts. Cell. 1991 Oct 18;67(2):303–310. doi: 10.1016/0092-8674(91)90182-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Smith C. W., Patton J. G., Nadal-Ginard B. Alternative splicing in the control of gene expression. Annu Rev Genet. 1989;23:527–577. doi: 10.1146/annurev.ge.23.120189.002523. [DOI] [PubMed] [Google Scholar]
  17. Stroeher V. L., Gaiser J. C., Garber R. L. Alternative RNA splicing that is spatially regulated: generation of transcripts from the Antennapedia gene of Drosophila melanogaster with different protein-coding regions. Mol Cell Biol. 1988 Oct;8(10):4143–4154. doi: 10.1128/mcb.8.10.4143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Tautz D., Pfeifle C. A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma. 1989 Aug;98(2):81–85. doi: 10.1007/BF00291041. [DOI] [PubMed] [Google Scholar]