Transcription in Isolated Nuclei

Maclean, Norman; Gregory, Stephen P.

doi:10.1016/b978-0-12-147608-3.50010-0

Cited by 5 publications

(2 citation statements)

References 207 publications

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“…run-on assays (e.g., see references 6, 19, 31, and review in reference 29) were to prove unfounded, it is clear that there is a significant difference in the transcriptional capacity of nuclei isolated from differentiating cells compared with nuclei from amebae. The fact that these differences reflect in vivo changes in mRNA concentration seems unlikely to be a coincidence.…”

Section: Discussionmentioning

confidence: 99%

Transcriptional regulation of coordinate changes in flagellar mRNAs during differentiation of Naegleria gruberi amebae into flagellates.

Lee

Walsh

1988

Mol. Cell. Biol.

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The nuclear run-on technique was used to measure the rate of transcription of flagellar genes during the differentiation of Naegleria gruberi amebae into flagellates. Synthesis of mRNAs for the axonemal proteins aand l-tubulin and flagellar calmodulin, as wel as a coordinately regulated poly(A)+ RNA that codes for an unidentified protein, showed transient increases averaging 22-fold. The rate of synthesis of two poly(A)+ RNAs common to amebae and flagellates was low until the transcription of the flagellar genes began to decline, at which time synthesis of the RNAs found in amebae increased 3-to 10-fold. The observed changes in the rate of transcription can account quantitatively for the 20-fold increase in flagellar mRNA concentration during the differentiation. The data for the flageilar calmodulin gene demonstrate transcriptional regulation for a nontubulin axonemal protein. The data also demonstrate at least two programs of transcriptional regulation during the differentiation and raise the intriguing possibility that some significant fraction of the nearly 200 different proteins of the flagellar axoneme is transcriptionally regulated during the 1 h it takes N. grubeni amebae to form visible flagella.Axonemes of eucaryotic flagella are composed of nearly 200 different proteins (28). Thus, neglecting proteins associated with basal bodies and the various rootlet structures found in most cells, the formation of cilia or flagella requires the synthesis and assembly of a minimum of 200 gene products. This complexity is particularly notable because some cells can assemble flagella quite rapidly.The best-studied example of flagellum formation is the biflagellated green alga Chlamydomonas reinhardtii. Most studies of flagellum formation in C. reinhardtii have taken advantage of the fact that the cells are readily deflagellated to examine the regeneration of flagella (e.g., see references 27, 32, 33, and 39). Regeneration involves both the assembly of preexisting flagellar proteins, which can produce about 50% of the original flagellar length, and the synthesis of new protein (33). Transient increases in a number of mRNAs coding for flagellar proteins have been documented during C. reinhardi flagellar regeneration (34), but changes in only two of these RNAs have been studied at the transcriptional level. Changes in the level of mRNAs for the a and ,B subunits of tubulin were found to be due, in part, to changes in the rate of their synthesis and, in part, to changes in tubulin mRNA stability (2,19). No data are available to suggest how proteins that are exclusively flagellar are regulated (C. reinhardtii contains only one form of ,-tubulin which presumably must be used in cytoplasmic and mitotic microtubules as well as flagella) (40). It is also of interest to ask whether the regulatory mechanisms governing regeneration of flagella in a normally flagellated cell would apply to a cell forming flagella de novo.The amebo-flagellate Naegleria gruberi provides an opportunity to examine this question. In contrast to C. rei...

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Section: Discussionmentioning

confidence: 99%

Transcriptional regulation of coordinate changes in flagellar mRNAs during differentiation of Naegleria gruberi amebae into flagellates.

Lee

Walsh

1988

Mol. Cell. Biol.

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“…Moreover, an exclusive nuclear staining was observed in Xenopus oocytes, including the follicle epithelial cells surrounding the oocytes, in epithelial cells of glands and ducts, fibroblasts and other dermal cells in the skin, cardiomyocytes, endothelial cells, and erythrocytes (our unpublished observations). Interestingly, nuclei of Xenopus erythrocytes that are reported to be inactive in transcription and replication (Maclean et al, 1973;Gregory et al, 1977;Maclean and Gregory, 1981;Coppock et al, 1989) were intensely stained by the antibodies specific for the 146-kDa protein (C and CЈ), indicating that the protein is a general nuclear constituent and that its presence does not depend on RNA or DNA synthesis. …”

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Molecular Characterization of a Novel, Widespread Nuclear Protein That Colocalizes with Spliceosome Components

Schmidt-Zachmann

Knecht

Krämer

1998

MBoC

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We report the identification and molecular characterization of a novel type of constitutive nuclear protein that is present in diverse vertebrate species, from Xenopus laevis to human. The cDNA-deduced amino acid sequence of the Xenopus protein defines a polypeptide of a calculated mass of 146.2 kDa and a isoelectric point of 6.8, with a conspicuous domain enriched in the dipeptide TP (threonine-proline) near its amino terminus. Immunolocalization studies in cultured cells and tissues sections of different origin revealed an exclusive nuclear localization of the protein. The protein is diffusely distributed in the nucleoplasm but concentrated in nuclear speckles, which represent a subnuclear compartment enriched in small nuclear ribonucleoprotein particles and other splicing factors, as confirmed by colocalization with certain splicing factors and Sm proteins. During mitosis, when transcription and splicing are downregulated, the protein is released from the nuclear speckles and transiently dispersed throughout the cytoplasm. Biochemical experiments have shown that the protein is recovered in a ϳ12S complex, and gel filtration studies confirm that the protein is part of a large particle. Immunoprecipitation and Western blot analysis of chromatographic fractions enriched in human U2 small nuclear ribonucleoprotein particles of distinct sizes (12S, 15S, and 17S), reflecting their variable association with splicing factors SF3a and SF3b, strongly suggests that the 146-kDa protein reported here is a constituent of the SF3b complex. INTRODUCTIONBiochemical fractionations and the use of antibodies to analyze the distribution of proteins in situ as well as recombinant DNA technologies have led to the identification of macromolecular domains within the mammalian cell nucleus. Beyond such obvious features as the nucleolus, heterochromatin, and the nuclear membrane, several particulate nuclear elements (termed "nuclear granules" or "nuclear dots") have been described that can be correlated with fundamental nuclear processes, e.g., transcription, RNA splicing, and processing of mature mRNA (reviewed by Spector, 1993).Splicing occurs in a multicomponent complex termed the spliceosome. Many of the detailed biochemical steps involved in the pre-mRNA splicing reaction have been extensively studied in vitro and are well understood (reviewed by Green, 1991;Moore et al., 1993). The major constituents of the spliceosome are the U1, U2, U4/U6, and U5 small nuclear ribonucleoprotein particles (snRNPs; reviewed by Baserga and Steitz, 1993). Moreover, spliceosomes are associated with numerous non-snRNP splicing factors, several of which have been purified and cloned (reviewed by Krämer, 1996;Will and Lü hrmann, 1997). Immunolocalization studies have revealed that proteins involved in pre-mRNA maturation tend to be heterogeneously distributed in the nucleus, suggesting that the processing reactions might be compartmentalized in vivo (Carter et al., 1993; however, see also Huang and Spector, 1996). In addition to the widespread nucleop...

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Developmental regulation of the alpha-mannosidase gene in Dictyostelium discoideum: control is at the level of transcription and is affected by cell density.

Schatzle¹,

Rathi²,

Clarke³

et al. 1991

Mol. Cell. Biol.

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In Dictyostelium discoideum, there is a group of genes that are expressed following starvation and when exponentially growing cells reach high densities. We have examined the expression of one of these genes, a-mannosidase. Using an a-mannosidase cDNA probe in Northern (RNA) blot analysis, we have shown that the previously observed increase in a-mannosidase enzyme-specific activity during development is due to an increase in the levels of a-mannosidase mRNA. mRNA levels reach a maximum by 8 h of development and then begin to decline by 14 to 22 h. Using nuclear run-on analysis, we have found that this gene is regulated at the level of transcription. We also examined the effects of cell-cell contacts, cyclic AMP levels, and protein synthesis on expression of this gene and found that they were not critical in regulating its expression. However, cell density did play a major role in the expression of a-mannosidase. High cell density or the presence of buffer conditioned by high-density cells was sufficient to induce expression of a-mannosidase, indicating that this is one of the prestarvation response genes. Finally, the a-mannosidase gene was not expressed in aggregationnegative mutant strain HMW 404.

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Transcription in Isolated Nuclei

Cited by 5 publications

References 207 publications

Transcriptional regulation of coordinate changes in flagellar mRNAs during differentiation of Naegleria gruberi amebae into flagellates.

Transcriptional regulation of coordinate changes in flagellar mRNAs during differentiation of Naegleria gruberi amebae into flagellates.

Molecular Characterization of a Novel, Widespread Nuclear Protein That Colocalizes with Spliceosome Components

Developmental regulation of the alpha-mannosidase gene in Dictyostelium discoideum: control is at the level of transcription and is affected by cell density.

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