Molecular genetics of Krüppel, a gene required for segmentation of the Drosophila embryo

Preiss, Anette; Rosenberg, Urs B.; Kienlin, Andrea; Seifert, E; Jäckle, Herbert

doi:10.1038/313027a0

Cited by 220 publications

(100 citation statements)

References 22 publications

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“…Further, to address the question of whether antisense RNA can inhibit genes transcribed in the cell nucleus, we injected antisense CAT RNA into the oocyte cytoplasm and then tested the expression of a plasmid DNA coding for CAT injected into the oocyte nucleus. Our data complement and support the findings of Melton who has independently obtained similar results using Xenopus globin mRNA (13), and the results of Preiss et al (16) who have shown that antisense RNA complementary to Kriipple mRNA can phenocopy a KrOpple mutation in Drosophila. …”

supporting

confidence: 82%

Translation of mRNA injected into Xenopus oocytes is specifically inhibited by antisense RNA.

Harland¹,

Weintraub²

1985

The Journal of Cell Biology

192

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The bacteriophage SP6 promoter and RNA polymerase were used to synthesize sense and antisense RNAs coding for the enzymes thymidine kinase (TK) and chloramphenicol acetyl transferase (CAT). Injection of antisense CAT RNA into frog oocytes inhibited expression of sense CAT mRNA. Similarly, antisense TK RNA inhibited expression of sense TK mRNA. Antisense RNAs were stable in oocytes and had no detectable effect on either the expression of endogenous proteins or on the expression of nonhomologous RNA transcripts. CAT activity expressed from a plasmid transcribed in the oocyte nucleus was also inhibited by antisense RNA injected into the oocyte cytoplasm. The data suggest that antisense RNA will be useful in identifying the function of specific mRNA sequences during early development of the frog.We are trying to identify and characterize genes that participate in normal embryonic development in the frog Xenopus laevis. An informative method of studying such genes would be to inhibit their expression in vivo to directly identify and assess the biological functions to which the genes contribute. While Xenopus embryos are a favorable system for experimental manipulations and biochemical analyses, the organism is not readily available to classical genetic analysis. One other way that genes may be inhibited is by the introduction of RNA complementary in sequence to the normal message (7). Hybridization between such an antisense RNA and the normal message may prevent translation or processing. It was previously shown (7,8) that the expression of exogenous genes as well as endogenous cellular genes can be dramatically and specifically inhibited when DNA molecules that express antisense RNA have been introduced into tissue culture cells.Injection of plasmid DNA directing the production of antisense RNA into Xenopus fertilized eggs is possible, however a high concentration of injected DNA is toxic to embryos (4, 19), and the available expression plasmids may not produce large enough amounts of RNA quickly enough to inhibit genes turned on in the rapidly dividing embryo. In contrast, injected RNA is not as toxic and, at least in the case ofglobin RNA, is very stable in embryos (5).Recently it has become possible to synthesize large amounts of a specific RNA from a plasmid containing a segment of cloned DNA downstream from the SP6 promoter (3,14). Using this technology, we have synthesized sense mRNAs coding for the bacterial enzyme chloramphenicol acetyl trans-1094 ferase (CAT) ~ and herpes simplex virus thymidine kinase (TK), as well as their antisense counterparts. We have first injected antisense, then sense mRNA into oocytes of Xenopus, and then extracted proteins for enzyme assay. Using these methods and quantitative measurements of sense and antisense RNA half-lives, we have determined (a) the extent to which expression of these sense mRNAs is inhibited at different concentrations of antisense RNA, and (b) whether an antisense RNA covering the full length of the protein-coding region of mRNA is necessary to eff...

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supporting

confidence: 82%

Translation of mRNA injected into Xenopus oocytes is specifically inhibited by antisense RNA.

Harland¹,

Weintraub²

1985

The Journal of Cell Biology

192

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“…Furthermore, one copy of the FX(10.7)-Kr-2F transgene rescues the segmentation phenotype of homozygous Kr 1 lack-of-function mutant embryos, indicating that FLAG-tagged Krü ppel is func-tional (34,35) (Fig. 1, C-F).…”

Section: Generation Of a Flag-tagged Krü Ppel Protein And Itsmentioning

confidence: 99%

“…To solve this apparent dilemma and to thereby demonstrate that our screen has indeed led to Krü ppel target genes, we asked whether Krü ppel does regulate ken expression in vivo by performing in situ hybridizations of ken probes to whole mount preparations of wild type and homozygous Kr 1 lack-of-function mutant embryos (34,35). In wild type, Krü ppel is initially expressed in a broad band in the central region of the blastoderm (15).…”

Section: Figmentioning

confidence: 99%

Genome-wide Mapping of in Vivo Targets of the Drosophila Transcription Factor Krüppel

Matyash

Chung

Jäckle

2004

Journal of Biological Chemistry

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Krü ppel (Kr), a member of the gap class of Drosophila segmentation genes, encodes a DNA binding zinc fingertype transcription factor. In addition to its segmentation function at the blastoderm stage, Krü ppel also plays a critical role in organ formation during later stages of embryogenesis. To systematically identify in vivo target genes of Krü ppel, we isolated DNA fragments from the Krü ppel-associated portion of chromatin and used them to find and map Krü ppel-dependent cis-acting regulatory sites in the Drosophila genome. We show that Krü ppel binding sites are not enriched in Krü ppel-associated chromatin and that the clustering of Krü ppel binding sites, as found in the cis-acting elements of Krü ppel-dependent segmentation genes used for in silico searches of Krü ppel target genes, is not a prerequisite for the in vivo binding of Krü ppel to its regulatory elements. Results obtained with the newly identified target gene ken and barbie (ken) indicate that Krü ppel represses transcription and thereby restricts the spatial expression pattern of ken during blastoderm and gastrulation.

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“…The name "Krüppel" is derived from the Drosophila protein krüppel (meaning "cripple" in German), which shares homology of proteins to DNA-binding domains 5,6) . Krüppel-deficient embryos in Drosophila die with consequences of abnormal thoracic and abdominal segmentation and thus appeared "crippled."…”

Section: Introductionmentioning

confidence: 99%

Role of Krüppel-Like Factor 4 and its Binding Proteins in Vascular Disease

Yoshida

Hayashi

2014

JAT

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Molecular genetics of Krüppel, a gene required for segmentation of the Drosophila embryo

Cited by 220 publications

References 22 publications

Translation of mRNA injected into Xenopus oocytes is specifically inhibited by antisense RNA.

Translation of mRNA injected into Xenopus oocytes is specifically inhibited by antisense RNA.

Genome-wide Mapping of in Vivo Targets of the Drosophila Transcription Factor Krüppel

Role of Krüppel-Like Factor 4 and its Binding Proteins in Vascular Disease

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