Ribosomal genes of the loach Misgurnus fossilis L. Isolation and restriction analysis

The ribosomal DNA of the teleost fish--loach has been isolated from sperm DNA by CsCl density gradient centrifugation. The rDNA sediments on density gradients by two heavy satellites beta = 1.715 and greater than 1.720. The DNA of the first satellite (1.715) was separated and treated by restrictases EcoRI and BamHI. It was shown that there are two EcoRI-sites in rDNA of loach, locating in 18S and 28S rRNA coding sequences. From tandem of repeating ribosomal genes EcoRI cuts out the fragment with homogeneous length-3 megadaltons (constant fragment) and heterogeneous population of fragments 11-13 megadaltons (major) and 7-8 megadaltons (minor fraction). The constant fragment contains mostly 28S coding sequence, and the heterogeneous fragment--18S coding sequence. The data indicate that the ribosomal genes of the loach as well as other higher eucaryotes were organized in genome as tandem of repeating units with heterogeneous length (10-16 megadaltons, 14.5-24 kb) due to heterogeneity of the length of nontranscribed spacer.     

Fine structure of pars neuro-intermedia of the loach,Misgurnus fossilis L.

Summary Two types of glandular cells are present in the pars intermedia of the loach, Misgurnus fossilis. Basophils are characterized by the presence in their cytoplasm of numerous secretory granules containing electron-dense and homogeneous material and by scarce endoplasmic reticulum. Weak acidophils contain in their cytoplasm abundant endoplasmic reticulum and numerous granules of different electron densities.The distal part of the neurohypophysis is composed of several types of neurosecretory axons, strongly branched pituicytes, numerous capillaries, and connective tissue elements. The axon terminals form the neuroglandular, neurovascular and neurointerstitial contacts. Some axon terminals are closely apposed to the basement membrane separating neurohypophysis from meta-adenohypophysis. At points of absence of continuity of this membrane, some neurosecretory axons become directly contiguous with cytoplasmic membranes of the intermedia cells.The investigation was partly supported by a research grant from the Zoological Committee of the Polish Academy of Sciences.     

Carboxylesterase-2 in the development of the loach (Misgurnus fossilis L.)

Two esterases splitting -naphthylacetate have been found in the tissues of adult loaches and in embryos. These were identified as arylesterase (E-1) (arylester hydrolase, E.C. 3.1.1.2) and carboxylesterase (E-2) (carboxylic ester hydrolase, E.C. 3.1.1.1.). In unfertilized loach eggs E-1 and E-2 synthesized during oogenesis were found. Active E-2 synthesized under the control of E-2 genes of the embryo appeared in embryos from the stage of 40–50 h of development. Maternal E-2 molecules synthesized in oogenesis or on the stored templates in embryogenesis persisted in larvae up to days 5–6 of development. Two genes controlling the synthesis of two forms of E-2 differing in electric mobility were found in the loach population from the delta of the Danube. The genes for fast and slow E-2 were shown to segregate in meiosis and to be allelic.     

Distant wave interaction in the early embryogenesis of the loach Misgurnus fossilis L

Groups of loach (Misgurnus fossilis L.) embryos of different ages were kept in different quartz cuvettes for 20-24 h so that only optic contact between the groups was possible. Subsequent observations showed that parameters of their development deviated from those in the control groups. Wave-mediated biocorrection proved to have both positive and negative effects, depending on the developmental stages of the interacting groups. Changes in spectral characteristics and polarization of biological radiation affected the results of the experiments. Various developmental abnormalities caused by distant wave-mediated interactions of embryos and specific to each combination of developmental stages and conditions of optic communication are described.     

Ultrastructural changes in loach (Misgurnus fossilis L.) embryos under borocin treatment

The ultrastructure of embryo cells of the loach (Misgurnus fossilis L.) at the stage of first division of blastomers in control and under the conditions of fluoroquinole borocin treatment has been investigated. The influence of this antibiotic at concentrations 5 and 25 mkg/ml has resulted in significant ultrastructural changes of embryo cells, such as hypertrophy of channels of the smoth and rough endoplasmic reticulum, disorganization of Golgy complex and mitochondrias, destruction of cytoplasm and mitochondrial membranes, rarefaction of cytoplasm and cell oedema. Such changes confirm the toxic influence of borocin on the embryo during early development.     

The primary structure of oocyte and somatic 5S rRNAs from the loach Misgurnus fossilis.

Somatic and oocyte 5S rRNAs from the liver and unfertilized eggs of the loach (Misgurnus fossilis have been sequenced and found to differ in six nucleotides. All the substitutions are confined to the 5'-half of the molecules; 4 of them are pyrimidine-pyrimidine substitutions, and 2 are purine-pyrimidine ones. Considerable differences, both in the position and the character of substitutions, have been established when these 5S rRNAs were compared with somatic and oocyte 5S rRNAs from Xenopus borealis and Xenopus laevis. Among the known primary structures, somatic 5S rRNA of M. fossilis is most similar to trout 5S rRNA.     

Effect of microinjections of cAMP-dependent protein kinase subunits on macromolecular syntheses in loach Misgurnus fossilis L. embryos

The level of cAMP and the effects of the regulatory and catalytic subunits of cAMP-dependent protein kinase of type II on protein, RNA and DNA synthesis in loach embryos were analysed. It was found that the injections of the catalytic subunit cause a sharp decrease in the rate of macromolecular synthesis while an increase in the concentration of this protein leads to the death of the embryo. Injections of the regulatory subunit result in marked stimulation of protein, RNA and DNA synthesis. The effect of the regulatory subunit on these processes is rather complex. Experiments on the transplantation of the nuclei from the embryos treated with the protein kinase subunits into normal embryos were carried out.     

The distribution of nuclear and cytoplasmic proteins in the blastoderm of the loach, Misgurnus fossilis L

The concentration of dry substance (protein) and the dry weight of nuclei, cytoplasm and cells from different blastoderm regions at the early blastula and midgastrula stages were determined by interferentional microscopy. It was shown that at the early blastula stage the dry weight of cells in the basal layer is higher than that in the outer layer. Although the protein concentration in the basal layer cells appears to be somewhat higher, differences in their dry weight are due primarily to the big volume of cytoplasm of the basal layer cells. By the midgastrula stage, the total (nucleus + cytoplasm) protein concentration increases (by 17% in the basal layer cells and by 9% in the outer layer cells) due to the increase of nuclear protein concentration. At the same time dry weight of these cells markedly decreases due to the decrease of their volumes in the process of cell divisions. At the midgastrula stage the epiblast cells have the highest dry weight due to the highest protein concentration in the cytoplasm and the biggest cell volume. The results obtained are discussed with respect to the data on the pattern of accumulation of newly synthesized protein in nuclei and cytoplasm with special reference to the duration of individual cell cycle phases.     

Foreign DNA in developing embryos of the loach 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.     

Molecular cloning and expression analysis of phospholipase Cdelta from mud loach, Misgurnus mizolepis

A gene encoding phosphoinositide-specific phospholipase C (PLC), designated ML-PLCδ, was cloned from mud loach (Misgurnus mizolepis) liver. A complete cDNA encoding ML-PLCδ was isolated by screening the cDNA library of mud loach liver and using the 5′-rapid amplification of cDNA ends (RACE) method. The full-length ML-PLCδ gene contains an open reading frame of 2325 base pairs encoding a 774 amino acid protein with a molecular mass of 88,072 Da; this corresponds to the size of the protein expressed in Escherichia coli BL21 (DE3) using pET28a vector. It contains all of the characteristic domains found in mammalian PLCδ isozymes (PH domain, EF-hands, X–Y catalytic region, and a C2 domain). A homology search revealed that ML-PLCδ shares relatively high sequence identity with mammalian PLCδ1 (51–52%) and catfish PLCδ (64%). The recombinant ML-PLCδ protein expressed as a histidine-tagged fusion protein in E. coli was purified to apparent homogeneity by Ni²⁺-NTA affinity chromatography. The recombinant ML-PLCδ showed a concentration-dependent PLC activity to phosphatidylinositol 4,5-bis-phosphate (PIP₂) and its activity was Ca²⁺-dependent, which was similar to mammalian PLCδ isozymes.     

Phospholipids and cholesterol in oocytes and embryos of the loach (Misgurnus fossilis)

The qualitative composition of lipid fractions in loach oocytes and embryos at different developmental stages is investigated by TLCh on silica gel. The variability in density of minor phospholipid fractions (lyzophosphatidyl choline, phosphatidylglycerine and cholesterol) is established in the period of synchronous divisions of blastomeres. The constant composition of major fraction of phospholipids in early embryogenesis of loach is registered.     

Isolation and properties of lactate dehydrogenase isoenzymes from loach (Misgurnus fossilis) skeletal muscles and eggs

The main lactate dehydrogenase (EC 1.1.1.27) isozyme from loach (Misgurnus fossilis) skeletal muscles was purified to homogeneity by polyacrylamide gel electrophoresis. The main group of lactate dehydrogenase isozymes which predominate in their activity in the unfertilized eggs of this teleost species and are stable to AgNO3 inhibition were partially purified. The effects of various concentrations of pyruvate, oxalate and urea on the activities of these purified enzyme preparations and their pH optima were studied. The antiserum for the purified lactate dehydrogenase isozyme from loach skeletal muscle was obtained. The decrease of the activity of this isozyme and that of the investigated group of isozymes from the eggs in the presence of increasing concentrations of antiserum was estimated.     

Morphogenetic consequences of partial removal of blastomere cytoplasm during early embryonic development of the loach, Misgurnus fossilis L.

The development of loach embryos is successfully regulated (normalized) after partial removal of the cytoplasm from one blastomere at the two- or four-cell stage or complete removal of one or two blastomeres at the stage of 8?C16 cells. Using time-lapse video imaging and morphometric analysis, it has been shown that this regulation is a two-stage process. At the first stage, the ratio between the volumes of the blastodisk and yolk sac is rapidly (within one or two cell cycles) restored almost to the initial level; at the second stage, morphogenesis of the embryo is modified according to its new structural features acquired after the operation. After several rounds of cytokinesis, the cytoplasm remaining in the operated blastomere fuses with the marginal yolk syncytium (periblast), which at the blastula stage forms a distinct extension at the operation site. This extension marks the site of embryonic shield formation. The results of morphometric analysis show that restoration of the initial blastoderm volume in operated embryos leads to a reduction of active tension at the blastoderm-yolk boundary and an increase in the ratio of blastoderm surface to its volume at the moment of epiboly initiation. As a result, the convergence of blastoderm cells to the operation site and the embryonic shield formation begin at a lesser degree of epiboly, compared to the control.     

Dramatically accelerated growth and extraordinary gigantism of transgenic mud loach Misgurnus mizolepis

Transgenic mud loaches (Misgurnus mizolepis), in which the entire transgene originated from the same species, have been generated by microinjecting the mud loach growth hormone (mlGH) gene fused to the mud loach -actin promoter. Out of 4,100 eggs injected, 7.5% fish derived from the injected eggs showed dramatically accelerated growth, with a maximum of 35-fold faster growth than their non-transgenic siblings. Many fast-growing transgenic individuals showed extraordinary gigantism: their body weight and total length (largest fish attained to 413g and 41.5cm) were larger and longer than even those of 12-year-old normal broodstock (maximum size reached to 89g and 28cm). Of 46 transgenic founders tested, 30 individuals transmitted the transgene to next generation with a wide range of germ-line transmission frequencies ranging from 2% to 33%. The growth performance of the subsequent generation (F₁) was also dramatically accelerated up to 35-fold, although the levels of enhanced growth were variable among transgenic lines. Three transgenic germ-lines up to F₄ were established, showing the expected Mendelian inheritance of the transgene. Expression of GH mRNA in many tissues was detected by RT-PCR analyses. The time required to attain marketable size (10g) in these transgenic lines was only 30–50 days after fertilization, while at least 6 months in non-transgenic fish. Besides growth enhancement, significantly improved feed-conversion efficiency up to 1.9-fold was also observed.     

Study of a cloned ribosomal operon region coding for 5.8S rRNA in the loach Misgurnus fossilis L

A fragment of the loach (Misgurnus fossilis L.) ribosomal operon containing 5.8S rDNA and adjacent regions of the internal transcribed spacer (ITS-1, and ITS-2) was sequenced. The 5'-terminal sequencing in 5.8S rDNA was corrected by analysing the primary structure of the loach 5.8S rRNA. This RNA was shown to be presented by three types of molecules; one of these was shorter by 4 nucleotides at the 5'-end because of the processing site being shifted in the rRNA precursor. The two other types differed in the 5'-terminal nucleotide (UMP or AMP). In the cloned fragment under study, the sequence of 5.8S rDNA has TMP at the 5'-terminus. The known nucleotide sequences of 5.8S rRNAs were compared in eukaryotes; as a result, conservative regions were revealed at the sites of molecule modification. All the 5.8S rRNAs of the vertebrates studied were found to have coincidences in the localization of nucleotide substitutions and other mutations (inversions and deletions). The authors propose a model for the secondary structure of ITS-1 and ITS-2 in the region of 5.8S rRNA processing.