RNA synthesis in isolated HeLa cell nuclei.

RNA synthesis has been studied in isolated nuclei of HeLa cells. The incubation medium has been optimized for RNA synthesis and the requirements for the presence of specific components previously used by other investigators has been examined. Nuclei isolated by centrifugation through 2 M sucrose synthesize RNA linearly for at least 1 h only at low temperature (25 degrees C). Low molecular weight RNA is found in the supernatant fraction after incubation; this RNA accounts for about 10% of the RNA synthesized. The RNA which remains within nuclei is of high molecular weight and processing of this RNA into molecules of the size of cytoplasmic mRNA does not seem to occur in isolated nuclei. We have studied the effect of an inhibitor of protein-nucleic acid interaction - aurintricarboxylic acid - on RNA synthesis by isolated nuclei. At concentrations below 0.1 mM, this drug does not inhibit RNA synthesis effectively, whereas at concentrations above 0.1 mM it inhibits RNA synthesis by about 80%. In view of the proposed mechanism of action of aurintricarboxylic acid, we suggest that completion of nucleotide chains initiated before nuclei isolation accounts for 20% of the RNA synthesized in our system by isolated nuclei, whereas nucleotide chains initiated during the in vitro incubation account for 80% of the RNA synthesized.     

DNA methylase from HeLa cell nuclei.

A DNA methylase has been purified 270-fold from HeLa cell nuclei by chromatography on DEAE-cellulose, phosphocellulose, and hydroxyapatite. The enzyme transfers methyl groups from S-adenosyl-L-methionine to cytosine residues in DNA. The sole product of the reaction has been identified as 5-methylcytosine. The enzyme is able to methylate homologous (HeLa) DNA, although to a lesser extent than heterologous DNA. This may be due to incomplete methylation of HeLa DNA synthesized in vivo. The HeLa enzyme can methylate single-stranded DNA, and does so to an extent three times greater than that of the corresponding double-stranded DNA. In single-stranded M. luteus DNA, at least 2.4% of the cytosine residues can be methylated in vitro by the enzyme. The enzyme also can methylate poly (dG-dC-dG-dC) and poly (dG, dC). Bilateral nearest neighbors to the 5-methylcytosine have been determined with M. luteus DNA in vitro and HeLa DNA in vivo. The 5' neighbor can be either G or C while the 3' neighbor is always G and this sequence is, thus, p(G/C)pmCpG.     

The Hough transform for locating cell nuclei.

A method of cytologic image segmentation that works purely on the basis of geometric information is described. Cell nuclei are assumed to be circular or elliptical. The latest ellipse-finding Hough transform is then used to locate the nuclei. The results are encouraging.     

Isolated HeLa cell nuclei synthesize meaningful DNA.

DNA replicated at the beginning of S phase was labelled by incubating nuclei isolated from cells arrested at the G/S border with radioactive deoxyribonucleoside triphosphate in a reaction mixture sustaining DNA synthesis. By hybridization against ribosomal RNA bound to nitrocellulose, the fraction of the labelled DNA which was complementary to rRNA could be quantified, and the stability of the RNA-DNA hybrids could be estimated by sequential elution of DNA at increasing temperatures. The results obtained indicate that the isolated nuclei make "meaningful" DNA, as judged by the melting characteristics of the hybrids between rRNA and the in vitro replicated DNA. Hybridization of the labelled DNA against rRNA fractionated by electrophoresis and blotted onto nitrocellulose verified the presence of sequences complementary to 18 S and 28 S rRNA.     

High-resolution cytometry of FISH dots in interphase cell nuclei.

BACKGROUND: Flow cytometry (FCM) and laser scanning cytometry (LSCM) provide indispensable tools for measuring large number of cells with low resolution. Confocal microscopy, on the other hand, is used for measuring small number of cells with high resolution. In this paper, we present a reasonable compromise between the two extremes. METHODS: We have developed a completely automated, high-resolution system (high-resolution cytometer, HRCM) capable of analyzing microscope slides with FISH-stained interphase nuclei in two dimensions as well as in three dimensions using a fully motorized epi-fluorescence microscope and a cooled digital CCD camera fully controlled by a high-performance computer which performs both acquisition and related on-line image analysis. The images of different dyes are acquired sequentially using highly specific filters and superimposed in computer memory. For each nucleus and each hybridization dot, user-selected attributes (such as position, size, intensity, etc.) are computed off-line using another processor or computer connected with a network. RESULTS: Using HRCM, it is possible to analyze multi-color preparations including UV-excited dyes as well as repeatedly hybridized preparations reacquiring individual nuclei. The speed of the acquisition and analysis is about 50 nuclei per minute in two dimensions and 1 nucleus per minute in three dimensions, but depends on the density of nuclei on the slide; the precision of the lateral and axial measurements is approximately 100 nm. CONCLUSIONS: Thus, using overnight acquisition, quantities comparable to those of FCM or LSCM measurements can be analyzed with an accuracy comparable to confocal microscopy. HRCM is suitable for a number of clinical and scientific tasks: routine diagnostics, follow-up of therapy, studies of chromatin structure, and many other different aspects of cell research.     

Protein synthesis in isolated cell nuclei

1. Nuclei prepared from calf thymus tissue in a sucrose medium actively incorporate labelled amino acids into their proteins. This is an aerobic process which is dependent on nuclear oxidative phosphorylation. 2. Evidence is presented to show that the uptake of amino acids represents nuclear protein synthesis. 3. The deoxyribonucleic acid of the nucleus plays a role in amino acid incorporation. Protein synthesis virtually ceases when the DNA is removed from the nucleus, and uptake resumes when the DNA is restored. 4. In the essential mechanism of amino acid incorporation, the role of the DNA can be filled by denatured or partially degraded DNA, by DNAs from other tissues, and even by RNA. Purine and pyrimidine bases, monoribonucleotides, and certain dinucleotides are unable to substitute for DNA in this system. 5. When the proteins of the nucleus are fractionated and classified according to their specific activities, one finds the histones to be relatively inert. The protein fraction most closely associated with the DNA has a very high activity. A readily extractable ribonucleoprotein complex is also extremely active, and it is tempting to speculate that this may be an intermediary in nucleocytoplasmic interaction. 6. The isolated nucleus can incorporate glycine into nucleic acid purines, and orotic acid into the pyrimidines of its RNA. Orotic acid uptake into nuclear RNA requires the presence of the DNA. 7. The synthesis of ribonucleic acid can be inhibited at any time by a benzimidazole riboside (DRB) (which also retards influenza virus multiplication (11)). 8. The incorporation of amino acids into nuclear proteins seems to require a preliminary activation of the nucleus. This can be inhibited by the same benzimidazole derivative (DRB) which interferes with RNA synthesis, provided that the inhibitor is present at the outset of the incubation. DRB added 30 minutes later has no effect on nuclear protein synthesis. These results suggest that the activation of the nucleus so that it actively incorporates amino acids into its proteins requires a preliminary synthesis of ribonucleic acid. 9. Together with earlier observations (27, 28) on the incorporation of amino acids by cytoplasmic particulates, these results show that protein synthesis can occur in both nucleus and cytoplasm.     

A DNA-activated protein kinase from HeLa cell nuclei.

A DNA-activated protein kinase (DNA-PK) was purified from nuclei of HeLa cells. Activity was associated with a single high-molecular-mass (approximately-300,000 Da) polypeptide when analyzed by gel filtration, denaturing polyacrylamide gel electrophoresis, and Western immunoblotting using a monoclonal antibody that also inhibits enzyme activity. Nuclear localization was indicated by subcellular fractionation and confirmed by immunofluorescence on whole cells. Double-stranded DNA stimulated phosphorylation of the 300-kDa polypeptide in purified preparations as well as phosphorylation of the exogenous substrates alpha-casein, simian virus 40 large T antigen, and the human heat shock protein hsp90. Autophosphorylation led to inactivation of the enzyme. The phosphorylation of casein was stimulated over 30-fold by DNA and was specific for serine and threonine residues. Bovine serum albumin and histone H1 were poor substrates for DNA-PK, and no phosphorylation of immunoglobulin G or histones other than H1 was observed. Supercoiled or heat-denatured DNA and synthetic double-stranded RNA or RNA-DNA copolymers did not stimulate casein phosphorylation by DNA-PK. Interaction of the enzyme with DNA in the absence of exogenous substrates was demonstrated by thermal inactivation and gel mobility shifts. These characteristics identify DNA-PK as distinct from other protein kinases described in the literature and suggest that activation by DNA is an important feature of the enzyme's in vivo function.     

Polyanion-induced release of polyribosomes from HeLa cell nuclei.

Intact detergent-washed HeLa nuclei contain a population of polyribisomes that were released by exposure to polyanions such as RNA or poly(U). The released material appeared by electron microscopic examination to be particles averaging about 200 to 300 angstroms in diameter. Sedimentation velocity analysis of the released particles indicated that the particles had S20,w values of 75 and 110. The particles stimulated amino acid incorporation in an ascites S-30 or S-100 extract at 2.5 mM Mg2+. Studies with a variety of antibiotics indicated that these polyribosomes were capable of elongating but not initiating protein synthesis. Although these polyribosomes may be of cytoplasmic origin, they appear unique in that agents thought to disperse chromatin are required for their release from the nucleus.     

The presence of intact mitochondrial DNA in HeLa cell nuclei.

Restriction analysis of DNA labelled with [32P]dCTP in an in vitro replication system with isolated nuclei from early S phase cells showed preferential labelling of restriction fragments derived from mitochondrial DNA (mtDNA) by a replication machinery distinct from that responsible for bulk nuclear DNA replication. Use of restriction nucleases with one recognition site in mtDNA gave rise to 16.5 kbp long fragments corresponding to full-length linearized mtDNA, indicating the presence of intact mtDNA in the isolated cell nuclei. Incorporation of dNTPs into mtDNA was not restricted to the S phase of the cell cycle. We were unable to increase the labelling of mtDNA by the addition of purified mitochondria or mtDNA to the nuclear replication system. These and other results presented is evidenced that the presence of mtDNA in the isolated nuclei was not due to uptake during preparation, thus indicating its presence in the cell nucleus in vivo.