L. de Jong

The RNA 3' cleavage factors CstF 64 kDa and CPSF 100 kDa are concentrated in nuclear domains closely associated with coiled bodies and newly synthesized RNA.

The cleavage stimulation factor (CstF), and the cleavage and polyadenylation specificity factor (CPSF) are necessary for 3′‐terminal processing of polyadenylated mRNAs. To study the distribution of 3′ cleavage factors in the nuclei of human T24 cells, monoclonal antibodies against the CstF 64 kDa subunit and against the CPSF 100 kDa subunit were used for immunofluorescent labelling. CstF 64 kDa and CPSF 100 kDa were distributed in a fibrogranular pattern in the nucleoplasm and, in addition, were concentrated in 1–4 bright foci. Double immunofluorescence labelling experiments revealed that the foci either overlapped with, or resided next to, a coiled body. Inhibition of transcription with alpha‐amanitin or 5,6‐dichloro‐beta‐D‐ribofuranosyl‐benzimidazole (DRB) resulted in the complete co‐localization of coiled bodies and foci containing 3′ cleavage factors. Electron microscopy on immunogold double‐labelled cells revealed that the foci represent compact spherical fibrous structures, we named ‘cleavage bodies’, intimately associated with coiled bodies. We found that approximately 20% of the cleavage bodies contained a high concentration of newly synthesized RNA, whereas coiled bodies were devoid of nascent RNA. Our results suggest that the cleavage bodies that contain RNA are those that are adjacent to a coiled body. These findings reveal a dynamic and transcription‐dependent interaction between different subnuclear domains, and suggest a relationship between coiled bodies and specific transcripts.

The protein composition of the nuclear matrix of murine P19 embryonal carcinoma cells is differentiation-stage dependent

The protein composition of the nuclear matrix of murine P19 embryonal carcinoma (EC) cells was compared with that of clonal derivatives of P19 EC differentiated in vitro, and with that of P19 EC cells induced to differentiate with retinoic acid (RA). Several major differences in nuclear matrix protein composition were found between the cell lines tested. Some polypeptides were found to occur only in EC cells, whereas others proved to be restricted to one or more of the differentiated derivatives. During RA treatment of EC cells a transient expression of some matrix proteins was observed. Several new proteins appeared, and others disappeared. Our data indicate that the protein composition of the nuclear matrix is a sensitive gauge for the differentiation state of cells.

Ultrastructural localization of active genes in nuclei of A431 cells

We have studied the ultrastructural localization of active genes in nuclei of the human epidermoid carcinoma cell line A431. Nascent RNA was labeled by incorporation of 5‐bromouridine 5′‐triphosphate, followed by pre‐embedment or postembedment immunogold labeling and electron microscopy using ultrasmall gold‐conjugated antibodies and silver enhancement. This combination of techniques allowed a sensitive and high resolution visualization of RNA synthesis in the nucleus. Transcription sites were identified as clusters of 3–20 gold particles and were found throughout the nucleoplasm. The clusters had a diameter of less than 200 nm. The distribution of clusters of gold particles in nuclei is preserved in nuclear matrix preparations. Nascent RNA is associated with fibrillar as well as with granular structures in the matrix. A431 nuclei contained on average about 10,000 clusters of gold particles. This means that each cluster represents transcription of probably one active gene or, at most, a few genes. Our study does not provide evidence for aggregation of active genes. We found transcription sites distributed predominantly on the surface of electron‐dense nuclear material, probably lumps of chromatin. This supports a model of transcription activation preferentially on the boundary between a chromosome domain and the interchromatin space.

Mapping of DNA replication sites in situ by fluorescence microscopy

Publisher Summary Sites of replication in S-phase nuclei can be visualized by the pulse labeling of nascent DNA. Fluorescence labeling techniques for nascent DNA allow more rapid and precise visualization of the sites of replication. These techniques are based on the incorporation of halogenated deoxynucleotide analogs into newly synthesized DNA, particularly bromo-, iodo-, or chlorodeoxyuridine (BrdU, IdU, and CldU, respectively) that are treated as deoxythymidine by the replication machinery. DNA that has incorporated such nucleotides can be fluorescently labeled by using primary antibodies that specifically recognize halogenated deoxyuridines, in combination with fluorochrome-conjugated secondary antibodies. Because halo-dU nucleotides can enter the cell by diffusion through the cell membrane, newly synthesized DNA can be labeled in vivo in living cells. A drawback of the use of halogenated nucleotides is that double-stranded DNA has to be denatured to allow antibodies access to the halo atoms. The rather harsh denaturation conditions may have a negative effect on the preservation of cellular and nuclear structure, even after the fixation of the cell. This may create problems if the labeling of replicating DNA is carried out in a dual-labeling protocol involving the immunocytochemical detection of another antigen.