Purifying nuclei.

Nuclear isolation methods exist since over 50 years and even today new procedures and amendments of standard methods are published. They can be classified into nonaqueous and aqueous methods. The latter can be subdivided into isotonic, hypertonic and hypotonic systems. In most cases the aqueous isolation renders nuclei closer to their physiological status in the cell. A standard method for the hypotonic isolation of nuclei is presented and the methodology of nuclear isolation is discussed.     

RNA transport in isolated myeloma nuclei. Transport from membrane- denuded nuclei

Nuclei prepared from MOPC-21 cells were treated with the nonionic detergents Triton X-100 or Nonidet P-40. Chemical analysis revealed that nearly 90% of the nuclear phospholipid was removed by detergent treatment. The membrane-denuded nuclei remained intact with preservation of nuclear pore complexes as demonstrated by electron microscopy. Ribonucleic acid transport from detergent-treated nuclei proceeded at the same rate and to the same extent as in control nuclei. Normal nuclear restriction of nucleic acids was unaltered by removal of the nuclear membranes. The effect of temperature on transport of RNA from freshly isolated myeloma nuclei with intact nuclear envelopes was studied. No temperature transition was associated with the transport process. These data indicate that the transport of macromolecules from isolated myeloma nuclei is independent of the nuclear membrane.     

Networks of neural nuclei

A new model of a neural system is presented. Neurons are considered to operate as parts of a neuronal ensemble called a neural nucleus, which has a homogeneous structure in connections among neurons. An excitatory nucleus shows one of the three fundamental properties in terms of the time course of the number of firing neurons in it: (i) increasing, (ii) constantly sustained and (iii) decreasing excitation. Mutually interacting structures of the neural nuclei are called nuclear networks. Simple nuclear networks consisting of one or a few nuclei are described to have functions such as integration, normalization and oscillation. In the nuclear networks inhibitory nuclei play an important role from the functional standpoint.     

Folding nuclei in proteins

When a protein folds or unfolds, it passes through many half-folded microstates. Only a few of them can accumulate and be seen experimentally, and this happens only when the folding (or unfolding) occurs far from the point of thermodynamic equilibrium between the native and denatured states. The universal features of folding, though, are observed just close to the equilibrium point. Here the 'two-state' transition proceeds without any accumulation of metastable intermediates, and only the transition state ('folding nucleus') is outlined by its key influence on the folding-unfolding kinetics. Our aim is to review recent experimental and theoretical studies of the folding nuclei.     

RNA metabolism in nuclei: selective transport of kappa exons from myeloma nuclei and adenoviral transcripts from infected HeLa nuclei.

Two independent systems and several analytical procedures have been used to establish that isolated mammalian nuclei selectively transport mature RNA polymerase I and II products. Murine myeloma nuclei retain physiologic restriction in our transport assay as assessed by the transport of the immunoglobulin kappa light chain mRNA and 18S and 28S rRNAs. Nearly 50% of the total kappa exons are transported as structurally intact mature mRNA molecules while less than 8% of either pulse-labeled or steady state kappa intron sequences are detected in the transported fraction. Ribosomal external transcribed spacer sequences also are absent in transported RNA. Release of cytoplasmic RNA from the outer nuclear membrane during the transport assay accounts for less than 10% of transported RNA. Nuclei isolated from adenovirus-infected HeLa cells at 20 hours post infection retain cellular actin mRNA and transport viral poly A+RNA. Ribosomal RNA is transported from infected nuclei although at a reduced rate compared to transport from mock-infected nuclei. Inhibition of transport of host mRNA is paralleled by the absence of pulse-labeled actin mRNA in the cytoplasm of infected cells. The implications of our transport data in relationship to intranuclear RNA trafficking are discussed.     

Cross-linking of proteins in nuclei and DNA-depleted nuclei from Friend erythroleukemia cells.

Reversible cross-linking of proteins in nuclei and DNA-depleted nuclei from Friend erythroleukemia cells was used as a probe to determine whether the protein structure was preserved following treatment with DNAase I. Interactions between histones were analyzed through cross-linking with 2-iminothiolane or dimethyl 3,3'-dithiobispropionimidate. No alterations in the interactions between intranucleosomal histone proteins resulted from digestion of the nuclear DNA. There was, however, a diminished extent of cross-linking of histone H1 to itself and to the intranucleosomal histones in DNA-depleted nuclei. The interactions of a group of nonhistone proteins with histone H3 could be monitored by cross-linking through the formation of disulfide bonds caused by oxidation of nuclei with H2O2. These interactions were not markedly affected by treatment of the nuclei with DNAase I. However, differences were observed in the extent of cross-linking of some of these proteins when cross-linking in nuclei from undifferentiated cells was compared to that in nuclei from cells which had been induced to differentiate with dimethylsulfoxide.     

Projections of the central cerebellar nuclei to the intralaminar thalamic nuclei in cats

Projections of the central cerebellar nuclei to the intralaminar thalamic nuclei were studied in cats with the use of light and electron microscopy. Almost all intralaminar nuclei were shown to obtain cerebello-thalamic projections. The entire complex of the central cerebellar nuclei serves as a source of such projections; yet, involvement of different nuclei is dissimilar. Destruction of the central and, especially, caudal regions of the fastigial nucleus evoked in the intralaminar thalamic nuclei degenerative changes in the nerve fibers (from swelling and development of varicosities up to total fragmentation). Pathological phenomena could be noticed in the most caudal regions of the above thalamic nuclear group, including the medial dorsal nucleus. Projections of the cerebellar interpositus nucleus were directed toward nearly the same regions of the intralaminar nuclei; degeneration was more intensive (covered thecentrum medianum) when posterior regions of the interpositus nucleus were destroyed. Destruction of the lateral cerebellar nucleus evoked a similar pattern of pathological changes, but degeneration was also observed in some structures of the ventral and anterior nuclear groups of the thalamus. Electron microscopic examination showed that degeneration of dark and light types developed in the fiber preterminals and terminals. It can be concluded that the central cerebellar nuclei project not only to the ventral complex of the thalamic nuclei, but also to the anterior, medial, and intralaminar nuclear groups (rostral and caudal portions).     

Isolation of skeletal muscle nuclei

A method for isolation of nuclei from rat skeletal muscle is described. The tissue is homogenized in the presence of Triton X-100, resulting in release of the nuclei from the muscle fibers. A crude nuclear pellet is prepared by differential centrifugation, and further purification is accomplished by centrifugation through dense sucrose. Bovine serum albumin is used to stabilize the nuclei during the isolation procedure. The isolated nuclei have been characterized microscopically and by chemical and enzymatic assay procedures.     

Stratification within centrifuged amoeba nuclei

Summary Two species of large, fresh water amoebae were ultracentrifuged and studied with the electron microscope. Emphasis was placed on the stratification of the nucleoplasm, including nucleoli, within the confines of the nuclear envelope during interphase. Three major strata were found in the nuclei of both amoeba species, namely the centripetal nucleoplasm, the middle chromatin stratum, and the centrifugal nucleolar mass. In the highly radioresistant A. proteus, the nucleolar mass separated into a centripetal electron-opaque layer and a centrifugal electron-lucent layer. The latter layer appears to be missing from the radiosensitive P. illinoisensis. The nature of these nucleolar layers and their possible relationship to differences in radiosensitivity between the two species of amoebae is discussed. The contents of the heavier of the two nucleolar layers in A. proteus might be resistant to radiation damage and may possess radiorestorative capacity.Work supported by U.S. Atomic Energy Commission. A part of the work was reported at the 17th Meeting of the Society of Protozoologists, Boulder, Colorado in 1964.     

Neutron diffraction of interphase nuclei

The neutron small-angle diffraction patterns obtained with native interphase nuclei are quite featureless and difficult to interpret. However, the difference between two patterns which have been obtained before and after having induced a change in the packing of the more stable structural entities, e.g. by a reduction of the salt concentration, shows significant changes at different scattering angles. These changes allow us to estimate the overall size of buffer-filled spaces which were formed by the separation of more stable structural entities and to establish accurate and non-destructive conditions at which changes of structure occur.Rat liver interphase nuclei have been prepared in a polyamine-containing buffer. The buffer was diluted from one-half down to one-fifth and one-tenth of its original salt concentration. In the first dilution step ($\text{1}\text{2}$ to $\text{1}\text{5}$), the difference spectrum was indicative of the formation of buffer-filled spaces with a most probable distance between a pair of scattering buffer molecules of 18 nm and with an overall size of the order of 45 to 50 nm. The second dilution step ($\text{1}\text{5}$ to $\text{1}\text{10}$) resulted in a strong increase of the intensity of the difference spectrum which had been observed in the first dilution step.     

Concealed nuclei in Saccharomyces strains

We have found that cells derived from heterokaryons (HK) showing phenotypical traits, coded by the nucleus of one parental strain and by the cytoplasm of the other, may produce mitotic progeny in which the second nucleus is apparently present but not expressed. This 'concealed' nucleus can be forced to expression after growth of these cytoductants on proper selective media. Using a micromanipulator, the buds containing both parental nuclei were isolated in various crosses. Cloning these HK from a rich medium (YEPD) revealed that nearly all of them were composed of a mixture of hybrid cells and cells of one of the parents. Cells of the other parent were present in a very small proportion, if detectable at all. We showed that the percentage of concealed HK decreases when limiting the growth of the strain that serves as a donor of the concealed nucleus. Consequently, our explanation for the presence of concealed nuclei in HK is the low production rate of daughter cells containing both nuclei, which accounts for the lack of a visible phenotype in HK, together with the low replication rate (or fast nuclease degradation) of one of the nuclei. In homosexual crosses, selection allows us to switch the concealed nucleus to normal replication rate. Some abnormalities of meiosis due to hidden nuclei are shown.     

Neutron scattering on nuclei.

Very small angle neutron scattering studies have been made on intact nuclei under a variety of solution conditions. Scattering maxima are observed at 30 to 40 nm and at 18 nm in most environments. Although the spacing, intensity and presence of the maximum near 40 nm varies considerably with environment the 18 nm is rather constant. The 30 to 40 nm maximum appears to be best interpreted by the presence of 35 to 50 nm diameter fibers in nuclei. An important result is that no scattering maximum was observed near 11 nm, suggesting that a tightly super coiled nucleofilament with such a pitch is not present.     

Spectrin-like proteins in plant nuclei

We analysed the presence and localization of spectrin-like proteins in nuclei of various plant tissues, using several anti-erythrocyte spectrin antibodies on isolated pea nuclei and nuclei in cells. Western blots of extracted purified pea nuclei show a cross-reactive pair of bands at 220-240 kDa, typical for human erythrocyte spectrin, and a prominent 60 kDa band. Immunolocalization by means of confocal laser scanning microscopy reveals spectrin-like proteins in distinct spots equally distributed in the nucleoplasm and over the nuclear periphery, independent of the origin of the anti-spectrin antibodies used. In some nuclei tracks of spectrin-like proteins are also observed. No signal is present in nucleoli. The amount and intensity of signal increases when nuclei were extracted, successively, with detergents, DNase I and RNase A, and high salt, indicating that the spectrin-like protein is associated with the nuclear matrix. The labelling is similar in nuclei of various plant tissues. These data are the first that show the presence and localization of spectrin-like epitopes in plant nuclei, where they may stabilize specific interchromatin domains.