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The dynamic nuclear redistribution of an hnRNP K-homologous protein during Drosophila embryo development and heat shock. Flexibility of transcription sites in vivo.

Buchenau P, Saumweber H, Arndt-Jovin DJ - J. Cell Biol. (1997)

Bottom Line: Injection of antibody into living embryos had no apparent deleterious effects on further development.The evaluation of two- and three-dimensional CLSM data sets demonstrated important differences in the localization of the protein in the nuclei of living compared to fixed embryos.These data are incompatible with a model of the interphase nucleus in which transcription complexes are associated with a rigid nuclear matrix.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.

ABSTRACT
The Drosophila protein Hrb57A has sequence homology to mammalian heterogenous nuclear ribonucleoprotein (hnRNP) K proteins. Its in vivo distribution has been studied at high resolution by confocal laser scanning microscopy (CLSM) in embryos injected with fluorescently labeled monoclonal antibody. Injection of antibody into living embryos had no apparent deleterious effects on further development. Furthermore, the antibody-protein complex could be observed for more than 7 cell cycles in vivo, revealing a dynamic redistribution from the nucleus to cytoplasm at each mitosis from blastoderm until hatching. The evaluation of two- and three-dimensional CLSM data sets demonstrated important differences in the localization of the protein in the nuclei of living compared to fixed embryos. The Hrb57A protein was recruited to the 93D locus upon heat shock and thus serves as an in vivo probe for the activity of the gene in diploid cells of the embryo. Observations during heat shock revealed considerable mobility within interphase nuclei of this transcription site. Furthermore, the reinitiation as well as the down regulation of transcriptional loci in vivo during the recovery from heat shock could be followed by the rapid redistribution of the hnRNP K during stress recovery. These data are incompatible with a model of the interphase nucleus in which transcription complexes are associated with a rigid nuclear matrix.

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Transcripts from the 93D  region and detection of the locus by  oligonucleotide hybridization. (A)  Schematic representation of the  transcripts from the heat shock  RNA-ω region in 93D. The gray  box indicates the location of the intron which is spliced out of the  smaller ω-c RNA. The repeat region of the long ω-n transcript is  shown in black. The sequence of  the fluorescently labeled oligonucleotide, Fl-P2, used as a probe for  ω-n is given below a 29 base transcript sequence which is strongly  conserved between the tandem repeats. B and C demonstrate that the  probe specifically binds to 93D  RNA in squashed polytene chromosomes. (B) DNA stained with  DAPI. (C) 93D hybridized with FlP2 under nondenaturing conditions.
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Figure 5: Transcripts from the 93D region and detection of the locus by oligonucleotide hybridization. (A) Schematic representation of the transcripts from the heat shock RNA-ω region in 93D. The gray box indicates the location of the intron which is spliced out of the smaller ω-c RNA. The repeat region of the long ω-n transcript is shown in black. The sequence of the fluorescently labeled oligonucleotide, Fl-P2, used as a probe for ω-n is given below a 29 base transcript sequence which is strongly conserved between the tandem repeats. B and C demonstrate that the probe specifically binds to 93D RNA in squashed polytene chromosomes. (B) DNA stained with DAPI. (C) 93D hybridized with FlP2 under nondenaturing conditions.

Mentions: A 29-base oligonucleotide with a sequence complementary to a repeated region in the 3′-end of the 93D transcript ω-n (Hovemann et al., 1986; Hogan et al., 1994) was synthesized and covalently labeled with fluorescein (Fig. 5 A). In squashed salivary gland chromosomes the labeled oligonucleotide hybridized exclusively to the 93D region both under conditions for DNA–DNA (data not shown) and for DNA–RNA hybridization (Fig. 5, B and C), demonstrating the specificity of the probe and the constitutive nature of the transcription from this locus. When formaldehyde fixed, whole-mount embryos were hybridized with the probe to RNA, i.e., under nondenaturing conditions, we detected one or two prominent spots per diploid nucleus with almost no labeling of the residual nuclear volume (Fig. 6 A). The polyploid nuclei of the embryo very often contained more than two hybridization spots, indicating that in these cases the loci were not synapsed. The fact that in all larval and embryonic tissues observed, the number of transcript accumulations as revealed by the hybridization parallels the number of expected chromosomal 93D loci leads us to conclude that within the resolution of the CLSM, our oligonucleotide probe reflects the location of nascent transcripts at the 93D locus. Therefore it can be used as a marker for the spatial location of this transcriptionally active gene in developing embryos. Fig. 6 A shows a projection image of normal diploid nuclei from a region of a fixed embryo hybridized to RNA (nondenaturing conditions) with the ω-n probe and stained with mAb Q18 and the merged multicolor image. These images have been reconstructed from the maximal intensities of 15 confocal sections for each fluorescence channel. A comparison of the ω-n/93D signal (Fig. 6 A) and the corresponding Hrb57A protein distribution in the merged panel indicates clearly that the protein was constitutively associated with the transcripts at 93D in these nonheat shocked embryos. Fig. 6 B contains a higher magnification image of two nuclei from the field in Fig. 6 A. The shape and size of the hybridization signal correlated almost precisely with that of an intense Q18 antibody binding region. In many but not in all nuclei of nonstressed embryos, Hrb57A showed the highest concentration in the 93D region. To determine the relative amount of the protein associated with 93D we measured the fluorescence intensities of the anti-Hrb57A antibodies in whole nuclei and in the nuclear subregion defined by the ω-n probe. Scoring many nuclei in several embryos, we found 2–5% of the total nuclear Hrb57A protein was associated with the 93D locus in normally developing embryos.


The dynamic nuclear redistribution of an hnRNP K-homologous protein during Drosophila embryo development and heat shock. Flexibility of transcription sites in vivo.

Buchenau P, Saumweber H, Arndt-Jovin DJ - J. Cell Biol. (1997)

Transcripts from the 93D  region and detection of the locus by  oligonucleotide hybridization. (A)  Schematic representation of the  transcripts from the heat shock  RNA-ω region in 93D. The gray  box indicates the location of the intron which is spliced out of the  smaller ω-c RNA. The repeat region of the long ω-n transcript is  shown in black. The sequence of  the fluorescently labeled oligonucleotide, Fl-P2, used as a probe for  ω-n is given below a 29 base transcript sequence which is strongly  conserved between the tandem repeats. B and C demonstrate that the  probe specifically binds to 93D  RNA in squashed polytene chromosomes. (B) DNA stained with  DAPI. (C) 93D hybridized with FlP2 under nondenaturing conditions.
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Related In: Results  -  Collection

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Figure 5: Transcripts from the 93D region and detection of the locus by oligonucleotide hybridization. (A) Schematic representation of the transcripts from the heat shock RNA-ω region in 93D. The gray box indicates the location of the intron which is spliced out of the smaller ω-c RNA. The repeat region of the long ω-n transcript is shown in black. The sequence of the fluorescently labeled oligonucleotide, Fl-P2, used as a probe for ω-n is given below a 29 base transcript sequence which is strongly conserved between the tandem repeats. B and C demonstrate that the probe specifically binds to 93D RNA in squashed polytene chromosomes. (B) DNA stained with DAPI. (C) 93D hybridized with FlP2 under nondenaturing conditions.
Mentions: A 29-base oligonucleotide with a sequence complementary to a repeated region in the 3′-end of the 93D transcript ω-n (Hovemann et al., 1986; Hogan et al., 1994) was synthesized and covalently labeled with fluorescein (Fig. 5 A). In squashed salivary gland chromosomes the labeled oligonucleotide hybridized exclusively to the 93D region both under conditions for DNA–DNA (data not shown) and for DNA–RNA hybridization (Fig. 5, B and C), demonstrating the specificity of the probe and the constitutive nature of the transcription from this locus. When formaldehyde fixed, whole-mount embryos were hybridized with the probe to RNA, i.e., under nondenaturing conditions, we detected one or two prominent spots per diploid nucleus with almost no labeling of the residual nuclear volume (Fig. 6 A). The polyploid nuclei of the embryo very often contained more than two hybridization spots, indicating that in these cases the loci were not synapsed. The fact that in all larval and embryonic tissues observed, the number of transcript accumulations as revealed by the hybridization parallels the number of expected chromosomal 93D loci leads us to conclude that within the resolution of the CLSM, our oligonucleotide probe reflects the location of nascent transcripts at the 93D locus. Therefore it can be used as a marker for the spatial location of this transcriptionally active gene in developing embryos. Fig. 6 A shows a projection image of normal diploid nuclei from a region of a fixed embryo hybridized to RNA (nondenaturing conditions) with the ω-n probe and stained with mAb Q18 and the merged multicolor image. These images have been reconstructed from the maximal intensities of 15 confocal sections for each fluorescence channel. A comparison of the ω-n/93D signal (Fig. 6 A) and the corresponding Hrb57A protein distribution in the merged panel indicates clearly that the protein was constitutively associated with the transcripts at 93D in these nonheat shocked embryos. Fig. 6 B contains a higher magnification image of two nuclei from the field in Fig. 6 A. The shape and size of the hybridization signal correlated almost precisely with that of an intense Q18 antibody binding region. In many but not in all nuclei of nonstressed embryos, Hrb57A showed the highest concentration in the 93D region. To determine the relative amount of the protein associated with 93D we measured the fluorescence intensities of the anti-Hrb57A antibodies in whole nuclei and in the nuclear subregion defined by the ω-n probe. Scoring many nuclei in several embryos, we found 2–5% of the total nuclear Hrb57A protein was associated with the 93D locus in normally developing embryos.

Bottom Line: Injection of antibody into living embryos had no apparent deleterious effects on further development.The evaluation of two- and three-dimensional CLSM data sets demonstrated important differences in the localization of the protein in the nuclei of living compared to fixed embryos.These data are incompatible with a model of the interphase nucleus in which transcription complexes are associated with a rigid nuclear matrix.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.

ABSTRACT
The Drosophila protein Hrb57A has sequence homology to mammalian heterogenous nuclear ribonucleoprotein (hnRNP) K proteins. Its in vivo distribution has been studied at high resolution by confocal laser scanning microscopy (CLSM) in embryos injected with fluorescently labeled monoclonal antibody. Injection of antibody into living embryos had no apparent deleterious effects on further development. Furthermore, the antibody-protein complex could be observed for more than 7 cell cycles in vivo, revealing a dynamic redistribution from the nucleus to cytoplasm at each mitosis from blastoderm until hatching. The evaluation of two- and three-dimensional CLSM data sets demonstrated important differences in the localization of the protein in the nuclei of living compared to fixed embryos. The Hrb57A protein was recruited to the 93D locus upon heat shock and thus serves as an in vivo probe for the activity of the gene in diploid cells of the embryo. Observations during heat shock revealed considerable mobility within interphase nuclei of this transcription site. Furthermore, the reinitiation as well as the down regulation of transcriptional loci in vivo during the recovery from heat shock could be followed by the rapid redistribution of the hnRNP K during stress recovery. These data are incompatible with a model of the interphase nucleus in which transcription complexes are associated with a rigid nuclear matrix.

Show MeSH
Related in: MedlinePlus