Tissue-specific trans-activation of the rabbit beta-globin promoter in Xenopus oocytes.
Identification of transcription factors regulating tissue-specific gene expression implies functional tests in transcription systems. In spite of its practical advantages, the Xenopus oocyte has only rarely been used for trans-activation studies, because some critical parameters inherent to the system may cause artefacts. Depending on the amount of DNA injected, even tissue-specific genes may be spontaneously transcribed. To develop a reliable trans-activation assay, we used the erythroid-specific rabbit beta-globin gene and, for comparison, the constitutively transcribed viral thymidine kinase gene. The viral gene is active over a wide range of injected DNA (0.2-10 ng), and addition of nuclear proteins from various cell types does not stimulate but often inhibits this activity. When large amounts of DNA are injected (greater than 10 ng), transcription is inhibited by self competition. Addition of nuclear proteins now re-establishes activity probably through increasing the pool of general transcription factors. By contrast, spontaneous activity of the beta-globin promoter occurs only within a narrow range of injected DNA (0.2-1 ng). At higher DNA concentrations (greater than 5 ng) spontaneous transcription becomes negligible. The addition of nuclear proteins from nonerythroid cells extracts has no or only a weak stimulatory effect on the beta-globin promoter. Only nuclear proteins isolated from erythroid tissues, bone marrow and spleen, bring about a strong transcriptional activation. Co-injection with either the polyoma virus, or the oviduct-specific chicken lysozyme gene shows that the beta-globin promoter is selectively activated by factors present in erythroid cell extracts.(ABSTRACT TRUNCATED AT 250 WORDS)
Rungger
D
D
Station de Zoologie expérimentale, University of Geneva, Chêne-Bougeries, Switzerland.
Muster
L
L
Boeck
R
R
Nichols
A
A
D000818:Animals; D003001:Cloning, Molecular; D005914:Globins / Q000235:genetics*; D008845:Microinjections; D009865:Oocytes / Q000502:physiology*; D009928:Organ Specificity / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011817:Rabbits; D015719:Single-Strand Specific DNA and RNA Endonucleases; D014158:Transcription, Genetic / Q000235:genetics; D015533:Transcriptional Activation / Q000502:physiology*
D005914:Globins; D015719:Single-Strand Specific DNA and RNA Endonucleases