Nuclear envelope associated protein that binds telomeric DNAs.

Rana temporaria oocytes at the 6th diplotene stage of maturation contain a special structure, the karyosphere capsule, with chromosomes covered and detached from the nuclear envelope (NE), though at the previous stage the telomeres were attached to the membrane, as characteristic of germ cells. The DNA-protein complexes from band shift assays with proteins extracted from oocyte NEs and telomeric DNA fragment (T(2)G(4))(130) were isolated and injected into a guinea pig. In the present paper the only protein of 70 kDa recognized by antibody (AB) in the NE is named the Membrane Telomere Binding Protein (MTBP). Western blots with guinea pig AB and AB against telobox peptide from TRF2 show that protein of 60 kDa (probably TRF1) belongs to the chromatin, but MTBP (TRF2 according to immunoprecipitation) belongs to the NE. In the somatic cell nuclei both proteins are present and recognized by AB against telobox peptide, but AB raised recognize only MTBP/TRF2 due to the epitope different from telobox. Combined in situ hybridization with the vertebrate telomeric DNA sequences (T(2)AG(3))(135) and immunocytochemistry with the MTBP AB showed them to be colocalized within the mouse nucleus. As it was shown by immunofluorescense of NE spread, MTBP is organized in a distinct pattern that looks like a network made of double-dots. Electron microscope immunogold staining with both ABs showed that the protein is localized on the outer surface of the oocyte NE within cup-like structures attached to the membrane. This is the first clear evidence of a protein, which could be responsible for the attachment of telomeres to the nuclear membrane.

Telomere-binding protein from the nuclear envelope of oocytes of the frog Rana temporaria.

The identification of telomere-binding protein on the nuclear envelope (NE) of the frog oocyte is reported here. Nuclei and the NE were isolated manually and extracted, and the extracts were used to search for DNA-protein specific interactions. Fragment of the Tetrahymena telomere sequence (T2G4)130 from the plasmid YAC4 was used as a labeled probe. DNA-protein complexes revealed by gel shift assay were cut out of the gel and injected into a guinea pig. The antibodies obtained have common antigenic determinants with keratins; this was shown on Western-blot under conditions of competitive binding. Antibodies were purified on an affinity column with keratins attached and used in the following work. Antibodies recognize one protein with molecular weight 70 kD among the NE proteins; no signal was obtained to proteins isolated from the inner part of the oocyte, but two polypeptides of 70 and 120 kD were detected in the proteins from frog liver nuclei. The 70-kD protein is situated on the NE in a distinct pattern that looks like a network, probably of double-dots as was shown by immunofluorescence of the NE. Electron-microscope immunogold staining showed that the protein is localized in the outer surface of the oocyte NE within cup-like structures attached to the membrane. Combined in situ hybridization using the mammalian telomeric DNA sequence (T2AG3)135 and immunocytochemistry using antibodies showed them to be colocalized in the frog blood cells, so telomere sequence coexists with the protein in the interphase nuclei. Most of the telomeres are fused in highly differentiated blood cells though a double dot signal is visible. The signal appears to be localized in the outer part of the nuclei. The existence of membrane telomere-binding protein allows a discussion of the attachment of the telemeters to the membrane.

Nuclear actin filaments and their topological changes in frog oocytes.

Previous morphological and biochemical studies have suggested that actin and actin-containing filaments (microfilaments) exist in the eukaryotic nucleus and that they perform important nuclear functions. However, the concept is not widely accepted. In this study, we demonstrate actin and bundles of actin in the nuclei of oocytes of Rana temporaria by immunoblotting and immunogold labeling/electron microscopy. The system and methods used here provided nuclei, free from cytoplasmic contamination. Additionally, we have compared the topological distribution of intranuclear actin filaments in two structurally and functionally distinct stages (stages 3 and 6) of oogenesis. The stage 3 nuclei are extremely active in rRNA transcription and contain multiple nucleoli located at the periphery with the central part occupied by the lampbrush chromosomes. The stage 6 nuclei are transcriptionally inert and contain both nucleoli and chromosomes confined to a small area in the central part. The nuclear lysates derived from the manually isolated stage 3 and 6 nuclei and the nuclear contents obtained by manually removing the nuclear envelope of stage 6 nucleus both contained actin as demonstrated by immunoblotting with an actin-specific monoclonal antibody. When examined by immunogold electron microscopy using the anti-actin antibody, the stage 3 oocyte nuclei showed distinct intranuclear tracks composed of bundles of actin that extended from the nucleoli and chromosomes to the nuclear envelope. The stage 6 oocyte nuclei, on the other hand, showed short stretches of actin bundles in the central part mainly in association with the nucleoli; none of these bundles extended to the nuclear envelope. Taken together, the above results suggest that actin is a structural component of the oocyte nucleus and that polymerized actin undergoes dramatic topological changes correlated with changes in the distribution of nuclear components and their function.

[Nuclear envelope of frog diplotene oocytes has telomere-binding activity]. / Iadernaia obolochka diplotennykh ootsitov liagushki obladaet telomersviazyvaiushchei aktivnost'iu.

A telomere-binding activity was found in the nuclear envelope of frog oocytes Rana temporaria by methods of binding on nitrocellulose filters and retardation. At present it is hardly possible to detect the proteins responsible for this activity. However, the results of this study allowed us to identify a specific mechanism of DNA-protein interaction, which provides the binding of the telomere to the envelope in meiotic cells.

[Medico-genetic study of the population of Kostroma Province. VI. The parameters of isolation by distance in the populations of the Bui and Shar'ya Districts of Kostroma Province]. / Mediko-geneticheskoe izuchenie naseleniia Kostromskoi oblasti. Soobshchenie VI. Parametry izoliatsii rasstoianiem v populiatsiiakh Buiskogo i Shar'inskogo raionov Kostromskoi oblasti.

A model of isolation by distance proposed by Malécot and developed by Morton is applied to the data on marriage distances collected in two regions of Kostroma Province. There is good agreement between the estimates of local inbreeding when using the isonymy method and the model of isolation by distance. Interpopulation kinship approaches 0 at the distance 700 km. The mean coefficient of kinship for parents in the families with autosomal-recessive pathology is 20 times higher than mean coefficient of kinship in the population.

[Effect of the temperature of oocyte maturation on the heat resistance of the body and cells in the progeny of the common frog]. / Izuchenie vliianiia temperatury sozrevaniia ootsitov na teploustoichivost' organizma i kletok potomstva u travianoi liagushki.

A study was made of the effect of low (4-5 degrees C) and high (17-22 degrees C) temperatures of oocyte maturation on different stages of development: a unicellular stage, early tadpole stage 39 and late tadpole, near the end of metamorphosis stage 52. Different temperatures in the course of oocyte maturation do not lead to reactive shifts in heat resistance of the organism and muscle cells of the progeny on either developmental stage examined.

[Correlation between the heat resistance of the provisional and definitive muscle tissue during the metamorphosis crisis in frogs]. / Korreliatsiia teploustoichivost'iu provizornoi i definitivnoi myshechnoi tkani v period metamorofnogo krizisa liagushek.

Using tadpoles of the lake frog Rana ridibunda Pall. during metamorphosis, a study was made of the heat resistance of the provisional muscle tissue of the tail and of that of two definitive muscles belonging to low-resistant (musculus iliofibularis) and high-resistant (musculus gastrocnemius) groups. It has been shown that during the late metamorphosis a statistically significant direct relation exists between the heat resistance of the provisional muscle tissue of the tail and definitive m. iliofibularis. A comparison with the earlier published data points to a positive correlation between each of these two characteristics and the survival of larvae at high injurious temperature.

[Medico-genetic study of the Uzbekistan population. X. Hereditary forms of hearing impairment in the Khorezm and Samarkand regions]. / Mediko-geneticheskoe izuchenie naseleniia Uzbekistana. Soobshchenie X. Nasledstvennye formy narushenii slukha v Khorezmskoi i Samarkandskoi oblastiakh.

Medical-genetic study in some populations of Khorezm (about 200,000) and Samarkand (about 56,000) provinces was performed with the view of identification of all family cases of deafness and deaf-mutism. 53 families with 140 affected patients were registered, in total. 8 nozological forms were found in this study, the most frequent being autosomal-recessive ones (6 nozological forms, 48 families, 122 patients). The autosomal-dominant forms were represented by 2 nozological forms (5 families with 18 patients). The reason for predominance of autosomal-recessive deafness in Uzbek population is discussed.

[Medico-genetical study of the Uzbekistan population. IX. Variability of hereditary pathology, territorial distribution of hereditary diseases and hereditary disease load in the population of the Urgut district of the Samarkand region]. / Mediko-geneticheskoe izuchenie naseleniia Uzbekistana. Soobshchenie IX. Raznoobrazie nasledstvennoi patologii, territorial'noe raspredelenie nasledstvennykh boleznei i otiagoshchennost' nasledstvennoi patologiei naseleniia Urgutskogo raiona Samarkandskoi oblasti.

Medical-genetic study was carried out in the population of Samarkand province (the population size about 150 000). Hereditary pathology was ascertained among families with two or more affected members with chronic diseases. 110 families with 210 patients were registered. The most frequent were autosomal-recessive disorders (42 nozological forms). 15 nozological forms are probably "new" conditions in this province, because they were absent in our previous medical-genetic study of this province. A tendency to local accumulation of families with the same disorder in small populations was observed. The load of autosomal-recessive disorders comprised 2.2 X 10(-3) affected, that of autosomal-dominant disorders being 0.51 X 10(-3) and of X-linked disorders being 0.25 X 10(-3) males. The importance of assortative maitings in manifestation of rare autosomal-recessive genes in Uzbek population is discussed.

[Effect of thermal selection of the male gametes on the resistance to temperature and chlorophos of the body and muscles in tadpoles of the common frog]. / Vliianie teplovogo otbora muzhskikh gamet na ustoichivost' k temperature i khlorofosu organizma i myshts golovastikov travianoi liagushki.

A study was made of the effect of thermal selection of parental spermatozoa on the heat resistance of the whole organism and of muscles in progeny of Rana temporaria, as well as of the organismal resistance to pesticides (0.0025 M chlorophos). The thermal selection of spermatozoa was performed by exposing their suspension to 36 degrees C for 30-40 minutes. With tadpoles, the resistance of muscles to 38 degrees C, was determined, in addition to the resistance of the organism to 34 degrees and to 0.0025 M chlorophos. The results of selection were stage--specific: at stages of growth (stages 39, 42, 50) the heat resistance of the organism was lower and that of muscles was higher than in controls. At the stage of metamorphosis proper (stage 52) the heat resistance of both the organism and muscles increased. The thermal selection of parental spermatozoa exerted no effect on the resistance of the progeny to 0.0025 M chlorophos.

[Bodily heat resistance and polymorphism of liver esterases of grass frogs]. / Teploustoichivost' organizma i polimorfizm ésteraz pecheni travianykh liagushek.

The determination of organismal heat resistance and qualitative composition of polymorphous liver esterases during heat acclimation (25 degrees) has been made on frogs Rana temporaria. During hybernation the most heat resistant frogs possess the homozygous allele of A2 esterase. Heat acclimation and the summer rise in temperature in nature lead to an increase in heat resistance of frogs and to the disappearance of selective advantage of animals possessing the isoenzyme of the A2A2 esterase. The functional homeostasis of populations can maintain biochemical polymorphism regardless of the selective advantage of individuals possessing one of the homozygous alleles of the isoenzyme.