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Purified mitochondrial DNA (mitDNA) from ovaries ofXenopus lae vis was injected into the nuclei (germinal vesicles) of large viteUogenic oocytes of the same organism and examined by electron microscopy ofthe spread nuclear contents. Normally located nuclei of untreated oocytes as weil as peripherally translocated nuclei of centrifuged oocytes were used. In addition, oocyte nuclei isolated and incubated under liquid paraffin oil were injected with DNA. The integrity oftranscriptional structures of endogenous chromosomal (Iampbrush chromosomes) and extrachromosomal (nucleoli) genes of the injected nuclei was demonstrated. Microinjected mitDN A was identified as circles of chromatin exhibiting polynucleosome-like organization and a me an contour length of 2.6 J.Lm, corresponding to a compaction ratio of the mitDN A of about 2 : I. This DNA packing ratio is similar to that observed after preparation of various kinds of native chromatin in low salt buffers. The chromatin circles formed from injected mitDNA only very rarely exhibited lateral fibrils suggestive of transcriptional activity. These results suggest that purified mitDNA can be transformed to normally structured chromatin when exposed to oocyte nuclear contents but is rarely , if at all , transcribed in this form and in this environment.
The structural organization of transcriptionally active DNA that contains cistrons for precursor molecules of ribosomal RNA is described in positively stained spread preparations from nuclei and nucleoli isolated from the green alga, Acetabularia mediterranea Lmx. These nuclei contain large aggregates of nucleolar subunits in which fibril-covered regions, the putative active cistrons for precursors of ribosomal RNA, alternate with fibril-free intercepts, the "spacers". The length distribution of the different intercepts of this DNA is given, and the pattern is compared with those shown in animal cell systems. The data are discussed in relation to problems of transcription and of amplification of ribosomal RNA genes.
The ultrastructure of th e growin g and ma turing primary nucleus of Acetabularia medite rranea and Acetabularia major has been studied with the use of various fi xation procedures. Particular interest has been focused on the deta ils of the nuclear periphery and the perinuclear region. It is demonstrated that early in nuclear grow th a characteristic perinucl ear structura l complex is formed which is, among the eukaryotic cells, unique to Acetabularia and re lated genera. This perinuclear system consists essentially of a) the nuclear envelope with a very hi gh pore frequency and various pore complex assoc iat ion s w ith granular and/or threadlike structures some of which are continuous with the nucleolus; b) an approx imate ly 100 nm thick intermediate zone densely filled with a filam entOus material and occasional sma ll membraneous structures from which the typical cytOplasmic and nuclear organe lles and particles are excl ud ed ; c) an adjacent Iacunar labyrinthum which is interrupted by many plasmatic junction channels between the intermed iate zone and the free cytOplasm; d) numerous dense perinuclear bodies in the juxtanuclear cytOplasm which a re especia lly frequent at the junction channels and reveal a composition of aggregated fibrillar and granul ar structures; e) very dense exclusively fibrill ar agg regates which occur either in assoc iation with t he perinuclear region of the lacunar labyrinthum or, somewhat further out, in the cytOplasmic strands between the bra nches of the lacun ar labyrinthum in the form of slender, characteristic rods or "sausages".
Several types of "irregular" structures in the arrangement of lateral fibrils were noted in electron microscopic preparations of transcriptionally active nucleolar chromatin from various plant and animal cells. Such forms include: I. Disproportionately long lateral fibrils which occur either as individual fibrils or in groups; 2. "Prelude complexes" and other arrangements of lateral fibrils in apparent spacer intercepts; 3. Thickening of the rDNA chromatin axis at the starting end of pre-rRNA matrix units; 4. Extremely long matrix units , the length of which exceeds that of the rDNA (double-strand) sequence complementary to the specific pre-rRN A (for abbreviations see text). In addition, the stability of high molecular weight RNAs contained in the nucleolar ribonucleoproteins during the preparation for electron microscopy was demonstrated by gel electrophoresis. The observations indicate that the morphological starting point of a pre-rRNA matrix unit is not necessarily identical with the initiation site for synthesis of pre-rRNA, but they rather suggest that the start of the transcriptional unit is located at least O.2-D.8 JLm before the matrix unit and that parts of the "apparent spacer" are transcribed. It is proposed that the pre-rRN A molecules do not represent the primary product of rDNA transcription but rather relatively stable intermediate products that have already been processed during transcription.
Some decades ago it was noted by cytologists that within the interphase nucleus large portions of the transcriptionally ("genetically," in their terms) inactive chromosomal material are contained in aggregates of condensed chromatin, the "chromocenters," whereas transcriptionally active regions of chromosomes appear in a more dispersed form and are less intensely stained with DNA-directed staining procedures (Heitz 1929, 1932, 1956; Bauer 1933). The hypothesis that condensed chromatin is usually characterized by very low or no transcriptional activity, and that transcription occurs in loosely packed forms of chromatin (including, in most cells, the nucleolar chromatin) has received support from studies of ultrathin sections in the electron microscope and from the numerous attempts to separate transcriptionally active from inactive chromatin biochemically (for references, see Anderson et al. 1975; Berkowitz and Doty 1975; Krieg and Wells 1976; Rickwood and Birnie 1976; Gottesfeld 1977). Electron microscopic autoradiography has revealed that sites of RNA synthesis are enriched in dispersed chromatin regions located at the margins of condensed chromatin (Fakan and Bernhard 1971, 1973; Bouteille et al. 1974; Bachellerie et al. 1975) and are characterized by the occurrence of distinct granular and fibrillar ribonucleoprotein (RNP) structures, such as perichromatin granules and fibrils. The discovery that, in most eukaryotic nuclei, major parts of the chromatin are organized in the form of nucleosomes (Olins and Olins 1974; Kornberg 1974; Baldwin et al. 1975) has raised the question whether the same nucleosomal packing of DNA is also present in transcriptionally active chromatin strands. Recent detailed examination of the morphology of active and inactive chromatin involving a diversity of electron microscopic methods, particularly the spreading technique by Miller and coworkers (Miller and Beatty 1969; Miller and Bakken 1972), has indicated that the DNA of some actively transcribed regions is not packed into nucleosomal particles but is present in a rather extended form within a relatively thin (4-7 nm) chromatin fiber.
Segregation of the nucleolar components is described in the differentiated nucleus of the generative cell in the growing Clivia and Lilium pollen tubes. This finding of a natural nucleolar segregation is discussed against the background of current views of the correlations of nucleolar morphology and transcriptional activity.