Characteristics of the ultrastructural organization of metaphase chromosomes in mouse embryos in the early cleavage stages

The structural organization of the mouse metaphase chromosomes in the early embryonic development (I-IV cleavages) was studied using serial ultrathin section. It was shown that in the first cleavage the metaphase chromosomes consist of DNP fibrils 20-25 nm in diameter, which are distributed nonuniformly along the chromosomes. It was suggested that parts of chromosomal arms formed by tightly packing DNP fibrils may correspond to the G-bands revealed by the routine Giemsa staining. In metaphase chromosomes of 8-16-cell embryos DNP fibrils form chromonema--thick threads about 90 nm in diameter. The chromonemata are evenly organized along chromosomal arms. The centromeric heterochromatin always consists of DNP fibrils tightly arranged in a block having no chromonemal level of organization. In all the cells studied chromosomes form structural contacts (associations) by their centromeric heterochromatin regions.     

Electron microscope examination of chromatin in hepatocyte nuclei within the first hourse after partial hepatectomy, II. The degree of chromatin condensation and the organization of fibrillar RNP components

The state of hepatocyte chromatin (the area occupied by the regions of condensed chromatin on ultrathin sections and the quantity of perichromatin RNP fibrils which was estimated by the area of the fibrillar zone and the concentration of fibrils within the same zone) were studied within the first hours after partial hepatectomy of guinea pigs. The area occupied by the regions of condensed chromatin on preparations with differentially revealed DNP and RNP components decreased by 12% in 2.5 hours since the operation had been performed, became normal in 5 hours, and again decreased by 30% in 9 hours. Decondensation of chromatin was accompanied with the increase of the number of perichromatin RNP fibrils, products of template activity of chromatin, and the rise of ethidium bromide binding. The binding of ethidium bromide by the chromatin of hepatocytes increased by 39% in 2.5 hours, returned to the control level in 5 hours and again increased by 22% in 9 hours.     

Intranuclear distribution of SV40 large T-antigen and transformation-related protein p53 in abortively infected cells

The intranuclear localization of SV40 T-antigen (T-Ag) and the cellular protein p53 was studied in SV40 abortively infected baby mouse kidney cells using two complementary methods of ultrastructural immunocytochemistry in combination with preferential staining of nuclear RNP components and electron microscope autoradiography. Both proteins were revealed in association with peri- and interchromatin RNP fibrils containing the newly synthesized hnRNA. In addition, T-Ag and p53 remained bound, at least in part, to the residual internal nuclear matrix following nuclease and salt extractions of infected cells. The localization of T-Ag was different in SV40 lytically infected monkey kidney cells since, in addition to hnRNP fibrils, the viral protein was also associated with cellular chromatin. However, when lytic infection was performed in conditions of blocked viral DNA replication, T-Ag was no longer associated with the cellular chromatin but remained bound to the hnRNP fibrils. We conclude that the transforming and lytic functions of T-Ag can be distinguished by different subnuclear distributions. The significance of the association of T-Ag and p53 with hnRNP fibrils and the internal nuclear matrix is discussed in relation to the role of these structures in the control of cellular mRNA biogenesis.     

Ultrastructural organization of polytene chromosomes of Chironomus thummi salivary glands]

An electronmicroscopical mapping of a number of regions of the polytene chromosomes of Ch. thummi salivary glands (3rd chromosome, right arm of the 1st chromosome, centromere regions, puffs 1-A2e, 1-A3ij, III-A5c and others) was done by the method of oriented ultrastructural sections of the unsquashed polytene chromosomes. The banding pattern on the electron micrograph was similar to the observed with the light microscope. The difference was that some doublets appeared as single cavity-containing bands with the double structure only in short regions under the electron microscope. It was also difficult to distinguish single bands in those regions where heavy adjacent bands were connected by dens, protrusions and anastomoses. These connections were most pronounced in the regions of the centromerers which had "spongy" appearance on the electron micrographs. These pictures may be connected with small interbands between heavy bands. Thin bands and some broad bands were frequently dotted. The puffs examined contained mainly RNP granules 200-400 A in diameter and RNP fibrils; BR-1 and BR-2 contained granules 500 A, RNP fibrils and smaller granules (200-400 A). BR and puffs were characterized by loop-like structures composed of granules arranged along the central DNP fibril. Only fibrils were presented in small interbands (0.05 mk), while larger interbands could include a small number of granules similar to those observed in puffs. It was found that centromere, telomeres and some heavy bands formed characteristic contacts with the nuclear membrane.     

Ribonucleoproteins containing mRNA in the cytoplasm of mouse plasmacytoma cells]

The rapidly labelled postribosomal ribonucleoprotein (RNP) found in the cytoplasm of mouse plasmacytoma cells were investigated. It has been shown that 45S and 80S particles contain relatively high molecular weight (approximately 12-17S) pulse-labelled RNA similar to the polyribosomal mRNA. No other postribosomal RNP was found which would contain an RNA with similar sedimentation characteristics. In CsC1 density gradients, the postribosomal RNP gives two peaks. One of them, the rapidly labelled component (rho 1.52 g/cm3) is found only in 45S RNP. The other rapidly labelled component (rho 1.36-1.41 g/cm3) is revealed in all investigated regions of sucrose gradients. The latter contains relatively low molecular weight RNA (approximately7-9S). These RNP are supposed to be informosome-like particles. The components with a buoyant density of 1.52 g/cm3 may represent an mRNP-45S subparticles complex. The rapidly labelled mRNA of 80S particles is released after EDTA treatment in the form of mRNP with a buoyant density of 1.45-1.47 g/cm3.     

Differential decondensation of mitotic chromosomes during the hypotonic treatment of cells as a possible cause of G-banding

Chinese hamster metaphase chromosomes were studied after treating the cells with a hypotonic 0.075 M KCl solution, routinely used for differential staining of chromosomes. After the incubation of cells in KCl for 5 seconds-40 minutes and fixation with glutaraldehyde, the chromosomes displayed DNP fibrils about 20 nm in diameter. These fibrils were unevenly packed along the chromosomal arms and formed sites of differential density. In sister chromatids, the even density sites were located symmetrically. The use of serial ultrathin sections of marker chromosomes (e.g., chromosomes with a telomeric disposition of nucleolar organizing regions) made it possible to establish a good correlation between the sites with the light packing of DNP-fibrils and G-bands, identified in the same chromosomes by the standard Giemsa-staining technique. Fixation of the KCl-incubated cells with the methanol: glacial acetic acid (3:1) solution did not change the DNP packing heterogeneity. The chromosomal banding state is reversible, for with the transfer of cells from KCl to the normal cultural medium the chromosomes become homogeneous in length. Thus, the data obtained allow to propose that the capacity of chromosomes for differential staining may be based on an uneven sensitivity of G- and R-bands to the decondensing effect of hypotonic treatment.     

SV40 recombinants carrying a functional RNA splice junction and polyadenylation site from the chromosomal mouse beta maj globin gene.

We have introduced a fragment of the chromosomal mouse beta major globin gene into SV40 and used the resultant hybrid virus to infect cultured monkey kidney cells. The mouse DNA fragment, which contains an intervening sequence and a poly(A) addition site, has been inserted in both possible orientations relative to the SV40 late region promoter. While the fragment is transcribed regardless of orientation, the RNA splice signal and poly(A) addition site are utilized only when the fragment is inserted in the "sense" orientation. Thus genomic mouse signals for both splicing and polyadenylation are recognized across species boundaries. Furthermore, since only an 18 bp segment was included on the 5' border of the intervening sequence, we can define a maximum length for this splice signal.     

Reconstitution of nucleoprotein complexes with mammalian heterogeneous nuclear ribonucleoprotein (hnRNP) core proteins

Newly transcribed heterogeneous nuclear RNA (hnRNA) in the eucaryote cell nucleus is bound by proteins, giving rise to large ribonucleoprotein (RNP) fibrils with an inherent substructure consisting largely of relatively homogeneous approximately 20-nm 30S particles, which contain core polypeptides of 34,000-38,000 mol wt. To determine whether this group of proteins was sufficient for the assembly of the native beaded nucleoprotein structure, we dissociated 30S hnRNP purified from mouse ascites cells into their component proteins and RNA by treatment with the ionic detergent sodium deoxycholate and then reconstituted this complex by addition of Triton X-100 to sequester the deoxycholate. Dissociation and reassembly were assayed by sucrose gradient centrifugation, monitoring UV absorbance, protein composition, and radiolabeled nucleic acid, and by electron microscopy. Endogenous RNA was digested and reassembly of RNP complexes carried out with equivalent amounts of exogenous RNA or single-stranded DNA. These complexes are composed exclusively of groups of n 30S subunits, as determined by sucrose gradient and electron microscope analysis, where n is the length of the added nucleic acid divided by the length of nucleic acid bound by one native 30S complex (about 1,000 nucleotides). When the nucleic acid: protein stoichiometry in the reconstitution mixture was varied, only complexes composed of 30S subunits were formed; excess protein or nucleic acid remained unbound. These results strongly suggest that core proteins determine the basic structural properties of 30S subunits and hence of hnRNP. In vitro construction of RNP complexes using model nucleic acid molecules should prove useful to the further study of the processing of mRNA.     

The dynamics of the structural reconstruction of mitotic chromosomes after their artificial decondensation in vitro

The treatment of isolated metaphase chromosomes with 5 mM Tris buffer caused their decondensation into DNP fibers 10 nm in diameter. The following increase in CaCl2 concentration induced the transition of nucleosomic DNP fibers into DNP fibers 20 nM and 40-50 nM in diameter, and the recovery of the whole chromosomes. However, in the similar conditions, the typical chromosomes (threads about 100 nm thick), chromomeres and G-bands were not reconstructed. According to these data, we assume that DNP threads 40-50 nm in diameter may be artificial (i.e. "pseudochromonemes"). The treatment of isolated chromosomes with 0.35 and 0.6 M NaCl prevents from formation of nucleomeric and pseudochromomeric fibers, although bodies of chromosomes can be recovered after the removal of HMG and H1 proteins. These observations point to a high stability of chromosomal fasteners providing the structural integrity of mitotic chromosomes.