[Extrachromosomal structures containing small nuclear RNP and coilin in the late vitellogenic oocytes of hibernating grass frogs]. / Ekstrakhromosomnye struktury, soderzhashchie malye iadernye RNP i koilin, v iadrakh pozdnikh vitellogennykh ootsitov zimuiushchikh travianykh liagushek.

We have studied extrachromosomal structures in the germinal vesicle (GV) of the late vitellogenic oocytes of hibernating frogs Rana temporaria. During this period of oogenesis, chromosomes are completely inactivated to be surrounded by a fibrillar karyosphere capsule (Gruzova, Parfenov, 1993). Using immunostaining and in situ nucleic acid hybridization, we have identified three types of extrachromosomal structures: Cajal bodies (CB), nucleoli, and micronucleoli. Immunostaining of GV spreads has revealed that CB and nucleoli contain coilin, a marker protein for CB. The nucleoli were also positively stained with antibodies against Sm-epitope and trimetylguanosine cap of small nuclear (sn) RNP. According to the results of in situ nucleic acid hybridization, the nucleoli contain U6 snRNA. To further investigate a distribution of coilin in GV of the late vitellogenic oocytes of R. temporaria, we injected myc-tagged transcripts of full length human coilin (Wu et al., 1994) into the cytoplasm of oocytes and followed the pathway of the newly translated protein with an antibody specific for the tag. Immunofluorescent staining of spread GV contents demonstrated a specific staining of the nucleoli within 3 h after injection. We suggest that the newly synthesized myc-coilin may be phosphorilated and targeted to the nucleoli.

[Intranuclear structures containing RNA maturation factors in early vitellogenic oocytes of Rana temporaria]. / Vnutriiadernye struktury, soderzhashchie faktory sozrevaniia RNK, v rannikh vitellogennykh ootsitakh travianoi liagushki.

We have examined the distribution of RNA processing factors in the germinal vesicle (GV) of the common frog Rana temporaria during early vitellogenesis by immunostaining on light- and electronmicroscopic levels and by in situ nucleic acid hybridization. Small nuclear RNPs (snRNP) and factor SC35 involved in pre-mRNA splicing occur in lampbrush chromosome loops and numerous granules 1-3 microns in size. These granules are identical to B snurposomes of Xenopus laevis and Notophtalmus viridescens described earlier (Wu et al., 1991). Some of B snurposomes are attached to homologous loops of lampbrush chromosomes. Immunofluorescent study of Cajal bodies/coiled bodies (CB) showed that sometimes CB have B snurposomes attached to their surface. In this case splicing factor SC35 is found in B snurposomes and B-like inclusions in CB matrix. In CB without attached B snurposomes splicing factor SC35 localizes throughout the whole organelle. Staining of GV spreads with antibodies against nucleolar protein NO38 revealed this protein in CB, nucleoli and micronucleoli. Using in situ nucleic acid hybridization and immunofluorescent staining we have found that on GV spreads from hibernating frogs B snurposomes contact nucleoli. Nucleoli contain snRNP. These data suggest that nucleoli may be storage sites of snRNPs during natural inactivation of RNA synthesis. During winter season in Rana temporaria GV nucleoli become compacted and a number of micronucleoli (less than 2 microns) dramatically increases. Analysis of micronucleoli showed that they contain rRNA, protein NO38, trace amount of U3 small nucleolar RNA and do not contain fibrillarin, involved as U3 in pre-rRNA processing. We suggest that decrease of rRNA synthesis during frog hibernation results in transformation of part of nucleoli in micronucleoli.

[The coiled bodies and satellite microbodies of the oocyte nuclei in hibernating Rana temporaria frogs contain actin]. / Skruchennye tel'tsa (coiled bodies) i mikrotel'tsa-satellity iader ootsitov zimuiushchikh liagushek Rana temporaria soderzhat aktin.

Immunocytochemical analysis of preparation of dispersed nuclei content in oocytes of III-IV stages of oogenesis, in terms of Dumont (1972), from hibernating grass frogs using monoclonal antibodies against actin, revealed two types of intranuclear structures containing this protein: coiled bodies (CB) and satellite microbodies (SM). Staining of these preparations with Rhodamin-phalloidin, known specifically to interact with fibrillar actin, did not reveal it in these structures. Results of our biochemical studies, using protease ESP32 specifically cutting only globular actin, are suggesting that both CB and SM contain globular actin. Gall et al. (1975) proposed that CB may be involved in assembling and sorting of small nuclear RNA for the three main RNA processing pathways: pre-mRNA splicing, pre-rRNA processing, and histone pre-mRNA 3'-end formation. Our finding of actin in CB allows a suggestion on actin involvement in the transport of RNA processing complexes from CB to some actual places where processing of RNA takes place. According to our previous data (Tsvetkov, Parfenov., 1994), SM participate in the karyosphere capsule formation. This process is preceded by SM fusion triggered presumably by actin.

[Spheres from the oocyte nuclei of the house cricket and the damselfly contain pre-mRNA splicing and pre-rRNA processing factors]. / Sfery iz iader ootsitov domovogo sverchka i strekozy-krasotki soderzhat faktory splaisinga pre-mRNK i protsessina pre-pRNK.

By the method of immunocytochemistry it has been shown that spheres from cricket and damselfly oocyte nuclei contain small nuclear RNA (snRNA) and fibrillarin, a protein involved in pre-rRNA processing. Besides, in cricket oocyte spheres coilin has been revealed, a protein which is part of intranuclear structures in somatic cells called coiled bodies. By the method of nucleic acid hybridization in situ, U1, U2 and U6 snRNAs were identified in spheres of cricket oocytes. Concentration of U2 snRNA was much higher compared to U1 and U6 snRNA. After a longtime (24h) incubation of cricket's ovary in the medium containing 3H-uridin, spheres remained unlabelled by contrast with a heavily labelled karyoplasm. Thus, the data obtained show a homology between spheres from the studied insects and coiled bodies of somatic cells, spheres from amphibian oocytes, and prenucleolar bodies in nuclei formed in vitro in Xenopus laevis egg extracts after adding DNA.

[The organization of dispersed nucleolar chromatin and the ultrastructure of the nucleoli in the oogenesis of the common frog]. / Organizatsiia dispergirovannogo nukleoliarnogo khromatina i ul'trastruktura iadryshek v oogeneze travianoi liagushki.

The structure of the nucleoli and their chromatin from grass frog Rana temporaria oocytes during different stages of oogenesis has been studied. Light microscopic analysis of dispersed according Callan's method (Callan et al., 1987) nucleoli of third stage of oogenesis (Dumont, 1972) revealed that by decreasing ionic strength to 25 mM spherical nucleoli got the appearance of necklace-like structures. Their electronmicroscopic analysis following by Miller's technique (Miller, Beatty, 1969) showed that they represent rosette-like structures which are connected to each other by ribosomal transcriptional units. Loops of the rosettes also are formed by transcriptional units of ribosomal genes. By the decreasing of transcriptional activity of nuclei from oocytes of 5-6th stages of oogenesis from hibernating frogs the structure of dispersed chromatin has been changed. The number of beads along the necklace-like structure axis decreased. Electron microscopic analysis of spread nucleolar chromatin from oocytes of these stages revealed the increase of part of inactive nucleosomal chromatin and the decrease of the number of loops in rosettes. The study of nucleoli ultrastructure showed that their morphology depends on their chromatin structure. In nucleoli with higher activity in oocytes of third stage of oogenesis fibrillar centers have not been found. At the same time in partially inactive nucleoli of 6th stage of oogenesis (Winter season) it was possible to identify well developed typical fibrillar centers. In those cases when nucleoli were completely inactive during the same stage of oogenesis and were not stained with 3H-actinomycin D, fibrillar centers were absent.

[The formation of the karyosphere in the oogenesis of insects and amphibians]. / Formirovanie kariosfery v oogeneze nekotorykh nasekomykh i amfibii.

This review deals with the authors' own and literary data on the ultrastructural and cytochemical organization of insect and amphibian oocyte nuclei, with special attention being paid to the karyosphere and its capsule. The evidence provided is supplemented with data on isolated karyospheres in Rana temporaria oocytes. A conclusion is made that the karyosphere is a complex structure which contains all chromosomes in the limited space of the oocyte nucleus, and that these chromosomes are, as a rule, in the process of inactivation. It is inferred that the karyosphere capsule commonly appears in gigantic oocyte nuclei (more than 100 micron in diameter) containing extrachromosomal DNA. The analogy of capsule organization in different invertebrate and vertebrate species is discussed, in addition to the involvement of presumably homologous nuclear structures (e.g. derivatives of synaptonemal complexes and nuclear envelope) in capsule formation. It is assumed that the karyosphere capsule is a specially organized part of the nuclear matrix. The capsule provides nuclear compartmentalization and chromosome localization in the germinal vesicle. Studies of this sort open up new possibilities to further investigation of intranuclear morphogenesis.

[Seasonal transformations in the lampbrush chromosomes and the morphogenesis of the karyosphere capsule in Rana temporaria oocytes detectable by an analysis of the isolated nuclear structures]. / Sezonnye preobrazovaniia khromosom-lampovykh shchetok i morfogenez kapsuly kariosfery v ootsitakh travianoi liagushki, vyiavliaemye pri analize vydelennykh iadernykh struktur.

A study was made of the seasonal changes in lampbrush chromosomes, accompanying structures and the karyosphere capsule, isolated from diplotene oocytes of Rana temporaria of stages 3-6 of oogenesis, as classified by Dumont (1972). The frogs were collected from their hibernation sites. The formation of fibrillar material of the karyosphere capsule was followed from stage to stage. Isolation of nuclei by hand, and dispersal of their content was carried out as described by Callan (Callan, 1986; Callan et al., 1987). A modified Miller's method (Miller, Beatty, 1969) was used for the ultrastructural analysis. The examination of lampbrush chromosomes shows that they change from season to season (summer, autumn, winter), reaching the maximum size in summer (Fig. 2). In autumn their transcriptional activity decreases, although their size remains unchanged. In this period each transcription unit of lateral loops contains about IO RNP-fibrils per 1 mkm, whereas in summer their number is 20 per 1 mkm (Fig. 4, 5). A large number (about 100 per each bivalent) of spheroidal granules about 1.8 mkm in diameter appear around the autumn bivalent (Fig. 3). They constantly accompany the chromosomes and therefore are called satellites. In winter, the satellites are of variable diameter (0.8-5.3 mkm) and still bound to lateral loops of chromosomes. The loops are notably reduced, and the association of the satellites shortened, to aggregate into knots.(ABSTRACT TRUNCATED AT 250 WORDS)

[The participation of endonuclease in the formation of extrachromosomal DNA and the possible mechanisms of the occurrence of gene amplification]. / Uchastie éndonukleazy v orbazovanii ékstrakhromosomnoi DNK i vozmozhnye mekhanizmy vozniknoveniia amplifikatsii genov.

We examined the extrachromosomal DNA (exDNA, Hirt fraction) in ethidium bromide sensitive and resistant cells of line L929. The exDNA amount is greater in the latter. The amount of exDNA in L929 cells makes 0.19% of the total cellular DNA; the exDNA amounts in cells, resistant to 5 and 50 micrograms/ml ethidium bromide are 0.22 and 0.33%, resp. Using labelling by BudR, it is shown that approximately 16% exDNA in L cells constituted amplified sequences to be excreting to the culture medium. The Zn-independent endogenous nuclease is activated in the resistant cells. The treatment with cycloheximide (50 micrograms/ml) resulted in the increase in the exDNA amount and in the activation of Zn-independent endonuclease. The data obtained suggested that the activation of Zn-independent endonuclease may lead to the increase in the exDNA amount and determine presumably a high rate of cell adaptability to environmental conditions.

[DNA from human lymphocytes contains a sequence, homologous to immunoglobulin gene kappa]. / DNK, vykhodiashchaia iz limfotsitov cheloveka, soderzhit posledovatel'nosti, gomologichnye immunoglobulinovomu genu kappa.

DNA released by human lymphocytes and hyman blood plasma DNA were examined by the electrophoresis, electron microscopy and Southern blot hybridization. The data obtained suggested that DNA released by lymphocytes contains covalently closed circular molecules. By the technique of the Southern blot analysis it was shown that DNA released by lymphocytes and human blood plasma DNA contain discretely sized molecules homologous to the C kappa fragment of the human Ig gene.

[Electron microscopy research on the chromatin structure of dispersed lampbrush chromosomes in the hen]. / Elektronno-mikroskopicheskoe issledovanie struktury khromatina v dispergirovannykh khromosomakh-lampovykh shchetkakh kuritsy.

The lampbrush chromosomes from the late previtellogenic and early vitellogenic oocytes of adult hens were spread according to Miller's technique and examined in electron microscope. The nucleosomes in the threads of the non-transcribed chromatin are uniformly distributed, the spaces between the nucleosomes correspond by their length to the linker DNA. Nucleosomes are absent in the transcriptional units with high intensity transcription. In transcriptional units with moderate and weak transcription the nucleosomes are identified in the axial chromatin fibril between RNA-polymerase granules, the space between them varying. The compactization ratio of DNA (the number of DNA mkm in a 1 mkm chromatin fiber) in non-transcribed fibrils is equal to 2.1, in transcriptional units with moderate and weak activity it is 1.7, and in transcriptional units with intensive transcription it is close to 1. The DNA compaction ratio in lateral loops of lampbrushes is determined by the intensity of transcription. It is supposed that in the native state the inactive chromatin with uniform spacing of nucleosomes has a supernucleosomal structure, while the DNA compaction ratio in the transcribed sequences corresponds to that on Miller's spreads.