Molecular cloning and characterization of ARS elements from the mud loach (Misgurnus mizolepis).
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Autonomously replicating sequences (ARSs) are thought to occur within, or adjacent to, the matrix attachment regions (MARs). To identify fish ARSs, MARs of the mud loach fish were obtained from nuclear matrices using a modified LIS method. These DNA fragments were screened for their ability to act as ARSs by being cloned into the ARS cloning vector, pURY19, and transformed into Saccharomyces cerevisiae. Sixteen ARSs were isolated, most of which were more efficient in transformation than the positive control vector, pURY19-2 microm, which contained the 2 microm circle origin of yeast. In particular, one clone, pURY19-ARS223, was 18 times more efficient in back-transforming E. coli than the positive control vector. Therefore, ARS223, which has strong ARS activity in yeast, could be a good candidate for inclusion in expression vehicles that are used to transfect fish cell lines or embryos. A DNA sequence analysis showed that the essential ARS elements contain potential ARS consensus sequences, and are predicted to have hairpin loop structures, or curved or kinked DNA. In addition, the MAR-Finder program suggested that ARSs also contain MAR motifs. These include AT tracts, ORI patterns, kinked DNA, ATC tracts, and Topoisomerase II consensus sequences. The in vitro matrix binding assay confirmed that all of the cloned ARSs could associate with the nuclear matrix. This indicates that ARSs elements may be located in or near the MARs. This is the first study that has identified and characterized ARSs in fish. (+info)
Effect of Misgurnus anguillicaudatus polysaccharide on immune responses of splenocytes in mice.
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AIM: To investigate the effect of Misgurnus anguillicaudatus polysaccharides (MAP) on immune responses of splenocytes in mice. METHODS: T lymphocyte proliferation (TLP) was measured by [3H]thymidine incorporation assay. Cytotoxic T lymphocyte (CTL) cytotoxicity and natural killer (NK) activity were determined by release of radioactive chromium [51Cr] from pre-labeled target cells. RESULTS: MAP 5 and 10 mg . kg-1 . d-1, ip for 7 d, could increase TLP, enhance the cytotoxicity of CTL and NK cells, and antagonize the effect of concanavalin A (ConA) on TLP suppressed by cyclophosphamide (CP). Inhibitory rates of CTL cytotoxicity were decreased from 51.4 % in CP control mice to 18.2 % and 35.1 % in MAP-treated CP mice, respectively. Furthermore, administration of MAP 10 and 20 mg . kg-1 . d-1, ip for 7 d, restored the reduced NK cell cytotoxicity caused by CP administration in mice. CONCLUSION: MAP has protective effect on augmenting T-cell-mediated immunity and NK activity in normal and CP-treated mice. (+info)
Cyclic changes of membrane conductivity in fertilized and activated eggs of teleost (Misgurnus fossilis) and their relation to the cell shape.
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Oscillations of membrane ionic conductivity, with a period similar to cell cycle duration, were observed in fertilized and activated loach (Misgurnus fossilis) eggs. In cleaving eggs the decrease in conductivity coincided with mitosis. Synchronously with the oscillations of membrane conductivity in activated as well as in fertilized eggs, rhythmic changes in blastodisc shape occurred. The blastodisc rounded up during the period of increasing membrane conductivity and flattened while conductivity decreased. Scanning microscopy of fertilized and activated eggs revealed differences in the surface relief of rounded and flattened blastodiscs. (+info)
Mitochondrial DNA content and mitochondrial gene transcriptional activities in the early development of loach and goldfish.
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The mitochondrial DNA (mtDNA) content of the mature eggs and embryos of loach and goldfish at early developmental stages were detected by means of dot hybridization. The transcription of mitochondrial cytochrome oxidase subunit I and II (COI and COII) genes during their early development was also detected by Northern hybridization. The experimental results showed that the mtDNA content of the mature egg as well as that of the embryos during the period from fertilized egg up to hatching stage in both fishes is maintained at a constant level, giving an average value of 7.40 x 107 molecules or 1.33 ng for every embryo in loach and an average value of 1.87 x 10(8) molecules or 3.31 ng for every embryo in goldfish. In both fish embryos, the COI and COII transcripts declined gradually after fertilization until late-blastula stage and then increased in early gastrula stage. This indicated that the transcription of mitochondrial genomes of these two freshwater fishes, which belong to different families, might be activated at the beginning of gastrulation. The steady-state amounts of mitochondrial messenger transcripts existing in the embryos during the early development in both fishes seemed to be regulated by both their half-lives and the transcriptional level of the mitochondrial genomes. The results showed that the transcription of the mitochondrial genome in the early developmental process in loach and goldfish was not regulated by a gene dosage mechanism. (+info)
The complete nucleotide sequence of the Crossostoma lacustre mitochondrial genome: conservation and variations among vertebrates.
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The complete mitochondrial (mt) genome of Crossostoma lacustre, a freshwater loach from mountain stream of Taiwan, has been cloned and sequenced. This fish mt genome, consisting of 16558 base-pairs, encodes genes for 13 proteins, two rRNAs, and 22 tRNAs, in addition to a regulatory sequence for replication and transcription (D-loop), is similar to those of the other vertebrates in both the order and orientation of these genes. The protein-coding and ribosomal RNA genes are highly homologous both in size and composition, to their counterparts in mammals, birds, amphibians, and invertebrates, and using essentially the same set of codons, including both the initiation and termination signals, and the tRNAs. Differences do exist, however, in the lengths and sequences of the D-loop regions, and in space between genes, which account for the variations in total lengths of the genomes. Our observations provide evidence for the first time for the conservation of genetic information in the fish mitochondrial genome, especially among the vertebrates. (+info)
Genetic relationship among gill-infecting Myxobolus species (Myxosporea) of cyprinids: molecular evidence of importance of tissue-specificity.
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The importance of tissue-specificity was studied in Myxobolus spp. infecting the gills of 7 cyprinid species. The 18S rDNA of 10 Myxobolus species was amplified by optimised nested-PCR, resulting in approximately 1600 bp PCR products. Phylogenetic trees generated by distance matrix and parsimony analyses revealed 4 main groups. Muscle-infecting species all belonged to the same group, while members of 2 gill-infecting groups were clearly distinguishable on the basis of tissue-specificity, and were also recognisable by differences in spore morphology. On the basis of tissue tropism, phylogenetic relationships among the species examined indicate that genetic separation is a more ancient evolutionary feature than host-specificity. (+info)
Dogmas and controversies in the handling of nitrogenous wastes: ammonia tolerance in the oriental weatherloach Misgurnus anguillicaudatus.
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The oriental weatherloach Misgurnus anguillicaudatus is an extremely ammonia-tolerant fish. Many ammonia-protection mechanisms have been reported in this fish. Six strategies used by this fish to deal with the problem of excess ammonia are described. The fish can (1) reduce ammonia production through reduction in protein and/or amino acid catabolism; (2) reduce ammonia production and obtain energy through partial amino acid catabolism leading to alanine formation; (3) detoxify ammonia to glutamine; (4) tolerate very high ammonia levels in its tissues; (5) get rid of ammonia as NH(3) gas and, probably, (6) possesses background K(+) channels that are impermeable to NH(4)(+). The effects of extracellular ammonia on the contraction performance of the heart from this fish were found to be the same as in rainbow trout, an ammonia-sensitive fish. It suggests that the hearts of most, if not all, fish species are protected against ammonia. MK-801, an NMDA receptor blocker, was found to have a protective effect against ammonia intoxication in the oriental weatherloach, which suggests that the NMDA receptor, as in mammals, is involved in ammonia toxicity. (+info)
From embryonic induction to cell lineages: revisiting old problems for modern study.
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A history of embryological studies of lens development and regeneration is sketched, paying special attention to the contribution of these studies to the conceptual aspect of embryology and later developmental biology. Emphasis is made on the fact that the interaction of different tissues during development, namely embryonic induction, was first discovered during studies of the lens and that the degree of the dependence of lens development on the inductive effect of the eye-cup varies among different species. Studies along the line of comparative-experimental embryology at the species level should be informative, in particular in combination with the recent trend of evo-devo studies. The processes of lens regeneration and in vitro transdifferentiation indicate the existence of multiple cell lineages with the potential of lens development in one animal. The occurrence of Wolffian lens regeneration in nature can be reconsidered from the new "eco-devo" viewpoint. (+info)