Studies on Isolated Nuclei

Maggio, Rachele; Siekevitz, Philip; Palade, George E.

doi:10.1083/jcb.18.2.267

Cited by 194 publications

(35 citation statements)

References 40 publications

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“…We found that this method exactly replicated the high nuclear purity initially reported. The cytoplasmic fraction was saved, and the nuclear fraction was subjected to a nucleolus:nucleoplasm separation protocol (Maggio et al 1963;Muramatsu et al 1963) modified for cultured mammalian cells by our laboratory Pederson 1972, 1973). Total RNA was then isolated from each fraction and analyzed by microarray profiling.…”

Section: Resultsmentioning

confidence: 99%

MicroRNAs with a nucleolar location

Politz¹,

Hogan²,

Pederson³

2009

RNA

168

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There is increasing evidence that noncoding RNAs play a functional role in the nucleus. We previously reported that the microRNA (miRNA), miR-206, is concentrated in the nucleolus of rat myoblasts, as well as in the cytoplasm as expected. Here we have extended this finding. We show by cell/nuclear fractionation followed by microarray analysis that a number of miRNAs can be detected within the nucleolus of rat myoblasts, some of which are significantly concentrated there. Pronounced nucleolar localization is a specific phenomenon since other miRNAs are present at only very low levels in the nucleolus and occur at much higher levels in the nucleoplasm and/or the cytoplasm. We have further characterized a subset of these miRNAs using RT-qPCR and in situ hybridization, and the results suggest that some miRNAs are present in the nucleolus in precursor form while others are present as mature species. Furthermore, we have found that these miRNAs are clustered in specific sites within the nucleolus that correspond to the classical granular component. One of these miRNAs is completely homologous to a portion of a snoRNA, suggesting that it may be processed from it. In contrast, the other nucleolar-concentrated miRNAs do not show homology with any annotated rat snoRNAs and thus appear to be present in the nucleolus for other reasons, such as modification/processing, or to play roles in the late stages of ribosome biosynthesis or in nonribosomal functions that have recently been ascribed to the granular component of the nucleolus.

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Section: Resultsmentioning

confidence: 99%

MicroRNAs with a nucleolar location

Politz¹,

Hogan²,

Pederson³

2009

RNA

168

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“…Previous work has already established that the structural features recognized in nuclei in situ are retained in nuclei isolated by aqueous sucrose methods (25,37,38) (see Fig. 1 a).…”

Section: Morphology Of Control and Triton X-io0 Treated Nucleimentioning

confidence: 97%

On the Attachment of the Nuclear Pore Complex

Aaronson

Blobel

1974

The Journal of Cell Biology

175

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Electron microscope examination of isolated rat liver nuclei after treatment with the detergent Triton X-100 revealed the complete removal of both the inner and outer membranes of the nuclear envelope. The envelope-denuded nuclei did not show any change in either shape or internal ultrastructure. Most strikingly, the nuclear pore complexes, which in untreated nuclei appear to be integral components of the nuclear envelope, were retained in their characteristic location at the distal ends of the channels leading through the peripheral heterochromatin.Determination of the chemical composition of detergent-treated nuclei showed that over 95% of the nuclear phospholipid was solubilized, thus corroborating the morphological absence of nuclear membranes. Furthermore, detergent treatment also solubilized approximately 10% of the nuclear protein. Analysis of the solubilized protein by polyacrylamide gel electrophoresis in the presence of SDS indicated that these proteins belong to a few specific classes which presumably represent the major polypeptides of the nuclear membranes.The total absence of the nuclear envelope on both morphological and biochemical grounds supports the idea that the nuclear pore complex does not require the membranes either for attachment to the nucleus or for maintenance of its own structural integrity. INTRODUCTIONIn eukaryotes, the nuclear pore complex is a ubiquitous organelle situated within the characteristic circular discontinuities ("pores") of the nuclear envelope (1-9). The roles which have been postulated for the pore and the complex fall primarily into two mutually compatible classes: (a) involvement in nucleo-cytoplasmic communication (9-12), and (b) organization of the interphase chromatin (13 16). Little direct evidence has been obtained to support either role, and there is only fragmentary information concerning the chemical composition of the complex (17, 18).Before attempting the isolation and chemical characterization of the pore complex, it was necessary to determine the structural relationships between the pore complex and its surrounding nuclear membranes and between the pore complex and the underlying peripheral chromatin.Nuclear pore complexes have been described in nuclear envelope fractions (19)(20)(21)(22)(23)(24)(25)(26). Their presence suggests that the bulk of the peripheral chromatin is not necessary for maintaining the gross structural integrity of the complex. However, significant amounts ofchromatin are routinely recovered 746

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“…Portions were taken for histological examination, and the rate of dihydrotestosterone formation was measured in 50 mg of slices as described above. Slices (25 mg) were also homogenized in duplicate for the measurement of protein (17), ribonucleic acid (21), and desoxyribonucleic acid (18) as described by Maggio, Siekevitz, and Palade (22).…”

Section: Methodsmentioning

confidence: 99%