[Foreign DNA in developing embryos of the loach Misgurnus Fossilis L]. / Chuzherodnaia DNK v razvivaiushchikhsia zarodyshakh v'iuna Misgurnus fossilis L.

The fate of pAT153 DNA microinjected into the embryos of loach was examined. At the earliest stages of development (2 h) the high molecular weight transgenome consisting of pAT153 sequences is formed. The transgenome replicates intensively during the course of early development (until the blastula--early gastrula stages), but later the replication slows down which results in the elimination of the transgenome from the majority of embryos at more advanced stages. The analyses of the transgenome structure with the help of restriction endonucleases have shown that, after microinjection of linear DNA, the high molecular weight transgenome is formed by stochastic ligation of the sticky ends of injected molecules. Our data suggest that the transgenome formation from circular or supercoil DNA occurs after its linearization by host endonuclease.

[Activation of RNA synthesis in loach embryos after injection of a nuclear extract obtained by using 0.35 M NaCl]. / Aktivatsiia sinteza RNK v zarodyshakh v'iuna posle in"ektsii ékstrakta iader, poluchennogo s pomoshch'iu 0.35 M NaCl.

An extract mainly containing chromatin nonhistone proteins was obtained by means of 0.35 M NaCl from nuclei isolated from loach (Misgurnus fossilis L.) embryos at the 18 hour developmental stage (late gastrula). Injection of a concentrated nuclear extract into the loach eggs was followed by intensified (1.5--2.0 fold) incorporation of radioactive precursors [3H]uridine or 14CO2 into RNA. The stage at which natural activation of the RNA synthesis occurs (6 hours, mid blastula) remains unchanged, but the rate of incorporation after the onset of synthesis activation (8 hours, late blastula) becomes greater.

[Distribution of solid liposomes on the surface of an epithelial cell layer]. / Raspredelenie tverdykh liposom po poverkhnosti plasta épitelial'nykh kletok.

A study was made of the adhesion of liposomes, composed of dipalmitoyl- or di-stearoylphosphatidycholine, on the surface of epithelial cells in culture. Sodium fluorescein was entrapped in liposomes for their visualization by fluorescence microscopy. It is found that sonicated unilamellar liposomes adhere predominantly along the sheet margins. Multilamellar liposomes and lipid-coated carmine particles adhere over the whole cellular surface. However, their adhesion along sheet margins was stronger, as evidenced by a brief trypsin treatment. A prolonged trypsin treatment removed all types of liposomes from the cell surface. After the cells were partly detached from each other, small liposomes readily adhered to the newly accessible cell margins. The existence of special lipid membrane-binding proteins on the cell surface is suggested.

[Focal contacts and the cytoskeleton]. / Fokal'nye kontakty i tsitoskelet.

Cultured cells attach to the substratum by means of specialized domains of cell surface, called focal contacts. The inner side of the cell membrane is associated in these structures with cytoskeletal elements, while the outer side is connected with extracellular matrix. The present review describes both light and electron microscopic methods of studying the focal contacts and ultrastructure of adhesion plaque, that is the cytoskeletal domain of focal contact. The proteins of adhesion plaque and focal contact membranes are also characterized. The processes of the formation of focal contacts and their association with the bundles of actin microfilaments in normal cultured fibroblasts are described in detail. Association of focal contacts with other cytoskeletal elements microtubules and intermediate filaments is discussed. The neoplastic transformation induced changes of focal contact system and cytoskeletal structures associated with contact sites are described.

[Competition of solid and fluid liposomes for binding and metabolism with lipids from the cell surface]. / Konkurentsiia "tverdykh" i "zhidkikh" liposom za sviazyvanie i obmen lipidami s kletochnoi poverkhnost'iu.

The competitive behavior of solid vs. fluid liposomes in liposome-cell adsorption and cell-to-liposome lipid transfer processes was investigated with L cells and FBT epithelial sheets. Binding and transfer experiments have demonstrated that: solid liposomes adhere to the cell surface as integral vesicles retaining the entrapped substance; fluid liposomes are partly disintegrated at the cell surface with concomitant entry of entrapped substances into the cytoplasm, while their lipids remain on the cell surface; fluid liposomes that escape lysis dissociate from the cell taking away cell lipid molecules. No lipid transfer occurs between the plasma membrane and solid liposomes. Cell-bound solid liposomes interfere with the transfer of cell lipids to fluid liposomes, while these in turn inhibit the binding of solid liposomes to the cell surface.

[Properties of inbred Drosophila melanogaster lines obtained from a population selected for an increased rate of embryonic development]. / Svoistva inbrednykh linii Drosophila melanogaster, poluchennykh iz populiatsii, selektirovannoi na povyshennuiu skorost' émbrional'nogo razvitiia.

Two heterogeneous Drosophila melanogaster populations were subjected to selection for an increased rate of embryonic development by picking out the first 10% of hatching larvae. After repeating this procedure in 15 generations, "fast" populations were obtained, in which the duration of embryonic development at high temperature (31-32 degrees C) was 30-40 min less than in nonselected control populations. The results of preliminary experiments on substituting the second and third chromosomes in the selected and control populations provide evidence that selected genes responsible for accelerated development are located on the second chromosome. Inbreeding in 12 generations of selected populations was used to obtain about 40 lines homozygous, in particular, at the alcohol dehydrogenase gene. In four lines, the developmental rate was higher than in a homozygous control line, but others did not differ from control or developed more slowly. The duration of embryonic development at 32 degrees C in fast lines was 50-70 min shorter than in control, but this difference was significantly less at lower temperatures (25 and 17 degrees C). Hence, high temperature is primarily a factor in providing conditions for the expression of genes determining the developmental rate, rather than a factor of selection for these genes. It is suggested that selected genes modify developmental rate dependence on temperature.

[Morphogenetic function of the nuclei in the early development of the common freshwater snail, Limnaea stagnalis]. / O morfogeneticheskoi funktsii iader v rannem razvitii bol'shogo prudovnika Limnaea stagnalis.

The early embryos L. stagnalis were placed in the actinomycin solution at the successive developmental stages. The permeability to actinomycin was previously increased by the pricking through egg capsules. The inactivation of nuclei by actinomycin up to the stage of 12 blastomeres resulted in the arrest of development at the 22 cell stage. The inactivation of nuclei at the subsequent development stages resulted in the developmental arrest at later stages. These data suggest that the embryonic development up to the 22 cell stage is provided by the nuclear function during oogenesis. The morphogenetic nuclear function of the embryo begins at the stage of 12 blastomeres and provides the embryonic development beyond the 22 cell stage.

[Nuclear function during early development of remote fish hybrids]. / Funktsiia iader v rannem razvitii otdalennykh gibridov ryb

The participation of paternal genome was studied in the development of remote hybrids obtained as a result of artificial insemination of the loach (Misgurnus fossilis) eggs by the sperm of aquarial Cyprinids (Brachydanio rerio, Danio malabaricus, Barbus tetrazona, Razbora heteromorpha, Carassius auratus) and Cobitids (Acanthophtalmus kuhlii). The hybrids obtained differed at the stage of hatching both from each other and from the loach by some morphological features. To study the function of heterologous nuclei, haploid nucleocytoplasmic hybrids were obtained by means of chromosome inactivation in the loach eggs by heavy doses of X-rays. The participation of paternal genome in development was estimated by comparison of the curves of viability of diploid and haploid hybrids with those of diploid, haploid and "anuclear" loach embryos. Patterns of mortality of embryos and larvae in each hybrid combination (percentage, stage) suggest the functioning of paternal genome already at the early stages of development. The activity of hybrid and heterologous nuclei was also estimated by the onset and the intensity of morphogenetic function which was determined by the time of embryonic death following nuclear inactivation at different stages. The onset of nuclear function in all hybrids coinsides with that in the loach, except B. rerio in which it occurs somewhat earlier. The data obtained prove the participation of paternal genes in development and maintenance of viability of embryos at all developmental stages beginning from the early ones (blastula).

[Does DNA alone determine the development of the organism? (the information aspect of the problem)]. / Tol'ko li DNK opredeliaet razvitie organizma? (informatsionnyi aspekt problemy).

Two closely related controversial problems are discussed: whether the developmental processes can be reduced to the synthesis of polypeptides encoded in DNA, and whether the information in DNA is equivalent to that in the adult organism. Critically considered are the ideas that DNA is only responsible for the protein synthesis, whereas morphogenesis proceeds independently and according to epigenetic regularities of its own. It is stated that development is the realization of genetic information in which more elementary (molecular) processes unambiguously determine a more complex cellular level which in its turn determines morphogenesis of tissues and organs. Various mechanisms of the appearance of new information in the course of development are considered. The statement is made that new information concerns only some individual characters of the organism, whereas most of information that determines the process of development and the structure of the adult organism is created in the course of evolution, is stored in DNA and inherited.

[The nature of the dependence of the rate of development on temperature determines the greatest efficiency of metabolism at optimal temperatures]. / Kharakter zavisimosti skorosti razvitiia ot temperatury opredeliaet naibol'shuiu éffektivnost' metabolizma pri optimal'nykh temperaturakh.

Metabolism intensity and development rate have different pattern of dependence on temperature. Oxygen intake and several other metabolic processes bear an exponential relationship to temperature. The pattern of this relationship is similar in different poikilothermal species. On the contrary, the relationship between the rate of development and temperature is species-specific and more complex. Hence, the curves obtained by plotting oxygen intake per developmental stage against temperature resemble parabola and the minimum values of oxygen intake correspond to optimal temperatures. Such a correspondence is almost solely determined by parameters of the relationship between the rate of development and temperature. Therefore, the efficiency of metabolism does not determine the range of optimal temperatures. It is suggested that adaptive alterations of this range during evolution proceed via changes of the parameters of relationship between the rate of development and temperature dependence. Relatively small number of mutations is required to produce such changes.

[Expression of genes controlling esterases during the embryonic development of hybrid fish]. / Ekspressiia genov, kontroliruiushchikh ésterazy v émbrional'nom razvitii gibridov ryb.

The electrophoretic mobility of several enzymes was studied in the embryos and early larvae of the hybrids between the loach (Misgurnus fossilis) and the aquarial cyprinids and cobitids (Acanthophthalmus). The cytosol aspartate aminotransferase is represented by one protein with the same mobility at all developmental stages both in the loach and in the hybrids. Malate dehydrogenase manifests four bands of isozymes which suffer no changes during the development. The electrophoretic profile of lactate dehydrogenase remains constant (10 isozymes) until hatching, but only 5 isozymes are found in the 5 days old larvae. Similar changes occur in the Misgurnus X Acanthophthalmus hybrids. The nonspecific esterases are represented by several proteins with different activities; their number increases after hatching. In the oocytes and adult specimens of Acanthophthalmus a characteristic and very active esterase was found which is absent in the loach. In the Misgurnus X Acanthophthalmus hybrids this esterase appears prior to the hatching and its activity later increases. Thus, the expression of the gene of one esterase begins in the end of embryogenesis.

[Cytophotometric and biochemical research on the amount of DNA in the nuclei of loach embryos injected with low-molecular RNA]. / Tsitofotometricheskoe i biokhimicheskoe issledovanie kolichestva DNK v iadrakh zarodyshei v'iuna, in''etsirovannykh nizkomolekuliarnymi RNK.

The cell cycle structure in the cells of loach embryos at the early blastula stage (5 h of development at 21 degrees) is markedly altered under the influence of injection of homologous low molecular weight nuclear RNA and, as a result, the number of cells in G2-phase. The DNA amount in the embryo increases by 20%. At the midblastula stage (7 h) no increase in the number of cells in G2-phase was found.

[Synthesis of ribosomal proteins in early development of the loach]. / Sintez ribosomnykh belkov v rannem razvitii v'iuna

The synthesis of ribosomal proteins (r-proteins) in the loach embryos was studied by the way of analysis of protein-labeled ribosomes isolated from the cytoplasm and purified from de novo synthesized polypeptide and contaminating proteins. The ribosomes at the mid-blastula and mid-gastrula stages were shown to contain no de novo synthesized proteins. The synthesis of r-proteins at the stage of organogenesis was revealed by sedimentation analysis of ribosomes in the gradient of sucrose concentrations, density analysis in the gradient of cesium chloride and analysis of the distribution of radioactivity in r-proteins separated from the labelled ribosomes by electrophoresis in polyacrilamide gel. At the stage of organogenesis, the exchange of ribosomal proteins with de novo synthesized r-proteins continues under the conditions of complete inhibition of RNA synthesis and incubation of unlabeled ribosomes with the labelled cytosol. The correlation between the absence of rRNA and r-protein syntheses at the early developmental stages and possible causes of the independence of r-protein synthesis and exchange from the simultaneous rRNA synthesis at the stage of organogenesis are discussed.

[Localization of aldolase activity in the embryos of loach]. / Lokalizatsiia al'dolaznoi aktivnosti v zarodyshakh v'iuna.

The activity of aldolase was determined in different parts of the loach (Misgurnus fossilis L.) embryo at the stages from the formation of axial organs till the beginning of embryonic movements (from 19 till 38 hrs of development at 21.5 degrees). In all parts of the embryo, the activity of aldolase at first decreased (21-23 hrs) and then increased. The region of somites is characterized by the highest absolute and specific activity at all developmental stages. The increase in the number of somites is accompanied by the fall and subsequent rise of aldolase activity. In the somites of different degree of differentiation, the enzyme activity changes in a similar way. Hence, there is no correlation between the morphological and biochemical differentiation of somites. Differences in the specific aldolase activity between the anterior and posterior halves of the embryo and the regions of head, somites and tail were found at the stages of 19-23 hrs of development. The maternal aldolase only is present at these stages, as was shown earlier. It means that the early stages of biochemical differentiation may be realized not by means of differential activation of genes controlling the enzyme, but by means of regylation of translation on the templates stored in oogenesis.

[Time of the action of genes controlling the activity of aldolase in embryonal development of groudling]. / Vremia deistviia genov, kontroliruiushchikh aktivnost' al'dolazy v embrional'nom razvitii v'iuna

The time of action of the genes controlling the decrease of aldolase activity (21-23 hrs of development) and its subsequent increase (23-36 hrs) was determined by means of inactivation of the nuclei by actinomycin or heavy doses of irradiation at succesive developmental stages. There exist two distinct periods of gene activity; the former (15-18 hrs) determines the rapid fall of maternal aldolase activity and the latter (21-27 hrs) its subsequent replacement by embryonic aldolase. This result is confirmed by the data concerning the changes in aldolase heat resistance in the hybrids of the loach and tropical cyprinids. The genes controlling the synthesis of the new aldolase and the morphogenesis which takes place at the same developmental stages are functioning at different times, i.e. the biochemical and morphological differentiations may occur relatively independently.

[The functioning of mammalian mitochondria injected into fish embryos]. / Funktsionirovanie mitokhondrii mlekopitaiushchikh, in''etsirovannykh v zarodyshi ryb.

The possibility of mammalian mitochondria functioning in fish embryos has been studied. Suspension of mitochondria isolated from the mouse fibroblast B-82/cap (chloramphenicol-resistant) and B-82 (chloramphenicol sensitive) cell cultures, were injected into the fertilized loach eggs. These embryos with an artificially increased number of mouse mitochondria developed and lived till the larval stages. Activity of cytochrome oxidase in these embryos was 1.5-2 times that in the control several hours after the injection, decreased during development and reached the control level by the gastrula stage. If these embryos with artificially increased number of mouse mitochondria were incubated in presence of chloramphenicol, only embryos that contained mitochondria from chloramphenicol-resistant cells survived, thus suggesting that the injected mitochondria do not degrade but are preserved and function in the cytoplasm of developing loach embryos.

[The temperature dependence of selection efficiency and the expression of the genes controlling the rate of embryonic development in Drosophila melanogaster]. / Temperaturnaia zavisimost' éffektivnosti selektsii i ékspressii genov, kontroliruiushchikh skorost' émbrional'nogo razvitiia u Drosophila melanogaster.

We have already shown that selection of heterogeneous D. melanogaster populations for the rate of embryogenesis at 32 degrees C may produce populations, in which 50% of the larvae are hatched 20-40 min earlier, i.e., 2-4% more rapidly than, in the control. Highly inbred strains were obtained from the selected populations and develop 5-7% more rapidly than the control populations. Here we studied separately the effects of temperature on the efficiency of selection and expression of the selected genotype. With this in view, multiple (9-15 rounds) selection of the first 10% of the larvae was performed at 17, 25, and 32 degrees C and the mean duration of embryogenesis (hatching of 50% larvae) at these three temperatures was determined in the entire selected population. Selection proved to be almost equally efficient at all temperatures. On the contrary, expression of the selected genotypes markedly depended on temperature: the rate of embryogenesis of the selected populations exceeded that in the control at 17 degrees C by 1.1%, at 25 degrees C by 2.5%, and at 32 degrees C by 3.5%. In the inbred strains this dependence was even more pronounced: 1.2%, 2.7%, and 5.7%, respectively. The temperature dependence of expression of the genes collected by selection and coding for accelerated development means that these genes affect the rate of development as the function of temperature by changing the angle of the curve inclination. Possible mechanisms of this phenomenon are discussed.

[Expression of paternal genes controlling cytochrome oxidase activity in hybrid fish]. / Ekspressiia ottsovskikh genov, kontroliruiushchikh aktivnost' tsitokhromoksidazy v razvitii gibridov ryb.

The heat resistance of the oxygen consumption by the mitochondria, temperature dependence of the Michaelis' constant (CM) and heat resistance of cytochrome oxidase were studied in the embryos and larvae of fish hybrids (Misgurnus X Brachydanio). The oxygen consumption by the mitochondria from the larvae of Misgurnus ceased (following the 10 min heating) at 50 degrees, from Brachydanio at 54 degrees and from the hybrids at 52 degress suggesting control of the respiratory function. CM of cytochrome oxidase has the same minimum in the larvae of Misgurnus and Brachydanio, therefore this criterion was not used to study the genetic control in their hybrids. The heat resistance of cytochrome oxidase (T50) differed in Misgurnus and Brachydanio and was of intermediate value in their hybrids. At the early stages of hybrid development T50 was of maternal type (Misgurnus) but beginning from the mid-gastrula stage T50 increased and attained the maximum prior to the hatching. Chloramphenicol did not affect the increase of T50 in hybrids, but actinomycin decreased it almost down to the level characteristic of Misgurnus. The data obtained suggest that the genetic control of cytochrome oxidase activity begins earlier than that of other studied enzymes.

[Heat resistance of aldolase of the hybrid embryos of sea urchins Strongylocentrotus droebachiensis and S, intermedius]. / Teploustoichivost' al'dolazy gibridnykh zarodyshei morskikh azhei Strongylocentrotus droebachiensis i S. intermedius

The time of expression of the genes controlling aldolase has been studied in the hybrid embryos female Strongylocentrotus droebachiensis X male S. intermedius. The enzyme heat resistance estimated by the temperature of 50% inactivation following the exposition for 30 min (T50) was used as its genetic marker. T50 of aldolase of the psychrophilic maternal species suffered practically no changes from the stage of mesenchyme blastula till the stage of 11 days old pluteus and equated 35.3 degrees. T50 of aldolase of autumn- and spring-spawning populations of the thermophilic paternal species equaled 39.5 degrees at the stage of 11 days old pluteus. The heat resistance of aldolase of the hybrid embryos did not differ reliably from that of maternal enzyme during the first 4 days of development (at 8 degrees) till the late gastrula stage and attained the maximum (T50 =36.9 degrees) on the 8th day (stage of pluteus). The expression of the genes controlling aldolase appears to take place between these developmental stages.

[Injection of mitochondria into oocytes and fertilized eggs]. / In'ektsiia mitokhondrii v ootsity i oplodotvorennye iaitsa.

The suspension of mitochondria isolated from the loach embryos or the frog heart were injected in the oocytes or fertilized eggs of the loach, newt, toad and frog in the amount roughly equivalent to the content of mitochondria in the egg. After the injection the oocytes did not differ during several days from the normal ones and the fertilized eggs of the loach, newt and South Afican clawed toad developed normally. The activity of cytochrome oxidase in the injected oocytes was kept at a somewhat higher level (1.4 to 1.9 vs 1.0 in the control) during several days. In the developing eggs the activity of cytochrome oxidase began to decrease from the blastula stage and attained rapidly the control level. The decrease of the enzyme activity is due to non-specific degradation of excessive mitochondria or to compensatory inactivation of the enzyme ensuring the maintenance of its normal activity during the development.