We developed a simple and inexpensive method to extract DNA from fresh and preserved fish scales.... more We developed a simple and inexpensive method to extract DNA from fresh and preserved fish scales. The procedure is based on boiling the scales in 5% Chelex 100, followed by digestion with proteinase K and subsequent absorption of genomic DNA using silica. A single fresh scale from larger species (e.g., tilapia) or a few scales from smaller species (e.g., 4 scales from zebrafish) provide over 200 ng of DNA, enough for at least 40 polymerase chain reaction amplifications. The procedure is applicable for DNA isolation not only from fresh and ethanol-preserved scales, but also from dried and formaldehyde-treated samples, and thus might be useful for investigating specimens stored in museums and other collections. Since the removal of a few scales is a gentle means of sample collection, this technique will allow analysis of genetic diversity, mating systems, and parentage in populations of endangered or ornamental fish with minimal experimental influence.
We isolated novel dinucleotide, trinucleotide, and tetranucleotide microsatellites from the genom... more We isolated novel dinucleotide, trinucleotide, and tetranucleotide microsatellites from the genome of Asian sea bass (Lates calcarifer). Two genomic DNA libraries were established, one was enriched for (CA)n repeats, while the other for (GATA)n, (GACA)n, and (AAC)n repeats. Sixty clones containing an insert between 250 and 1000 bp in size were sequenced from each library; altogether 50 (43%) of them contained microsatellites. Forty microsatellites were characterized in 16 unrelated Asian sea bass individuals. Twenty-eight of them (70%) showed specific amplification and polymorphism. The allele number per loci varied between 2 and 20 with an average of 5.3, while expected heterozygosity ranged from 0.31 to 0.95 with an average of 0.64. At some loci allele sizes spread over a wide range (>100 bp). No significant correlation (r = 0.23, df = 31, P > 0.05) was found between the repeat number and the number of alleles. A whole broodstock containing 170 individuals was analyzed by using 8 selected polymorphic microsatellites. The average number of alleles per locus was 11.8 (range, 4–21). The expected heterozygosity ranged from 0.57 to 0.90 with an average of 0.75, while the fixation index was 0.02. Genetic similarity between individuals ranged from 0 to 0.72. Comparison of allele frequencies between the broodstock and the 24 nonrelated individuals revealed some unique alleles.
In contrast to Ferguson plots based on relative mobilities, Ferguson plots of proteins in polyacr... more In contrast to Ferguson plots based on relative mobilities, Ferguson plots of proteins in polyacrylamide gel electrophoresis based on their absolute mobilities were found to be linear under unusual polymerization conditions which yield relatively wide gel fibers and a low total fiber length per unit weight, but not under previously and commonly used conditions. These linear Ferguson plots in gels of 1, 3 and 5% crosslinking intersect at a single gel concentration between 1 and 2 %T (M-point). It is postulated that the measure of free mobility of the proteins is the M-point, and not the intercept of their Ferguson plots with the mobility axis as assumed previously. This postulate abolishes the well-known paradoxical interpretation of the increase with %C of the linearly extrapolated intercept of the Ferguson plot with the log(mobility) axis (designated Yo) in terms of free mobility. The postulate is also compatible with the interpretation of the points of intersection of the Ferguson plots of oligomeric series of proteins at finite gel concentrations (designated μ-points) as their common free mobilities.
Investigations on paraquat toxicity in fishes. J Nemcsok, L Orban, B Asztalos, Z Buzas, A Nemeth,... more Investigations on paraquat toxicity in fishes. J Nemcsok, L Orban, B Asztalos, Z Buzas, A Nemeth, L Boross Water International 10:22, 79-81, 1985. The effect of paraquat in different concentrations on three fish species, carp ...
Liposome-mediated gene transfer was used to introduce large DNA constructs into zygotes of Africa... more Liposome-mediated gene transfer was used to introduce large DNA constructs into zygotes of African catfish. The technique is based on the delivery of recombinant bacteriophage lambda particles (or DNA-protamine complexes) into the cytoplasm of target cells by negatively charged, large unilamellar liposomes. Dechorionated zygotes and early fish embryos were treated with the transforming liposomes. Expression of the introduced reporter genes during the first three weeks of the development of the larvae was followed by measuring the activity of corresponding enzymes. These assays have indicated very efficient DNA uptake into the embryos.
... of them have shown quite promising preliminary results in various labs: sperm electroporation... more ... of them have shown quite promising preliminary results in various labs: sperm electroporation (Muller et al., 1992; Sin et al., 1993), electroporation of fertilized eggs (Inoueetal., 1990; Buono and Linser, 1991; Powers ... In: R. Billard and J. Marcel (Editors), Aquaculture ofCyprinids. ...
We have successfully performed interspecific androgenesis between two cyprinid species. Gamma-ray... more We have successfully performed interspecific androgenesis between two cyprinid species. Gamma-ray irradiated eggs of common carp were fertilized with fresh and cryopreserved sperm of three different goldfish varieties and the haploid embryos were then heat-...
The common carp is an important fish species satisfying ornamental, food and recreational fisheri... more The common carp is an important fish species satisfying ornamental, food and recreational fisheries’ needs worldwide, but in common with other cyprinid fishes, it is particularly renowned for its environmental tolerance. Investigating the mechanistic basis of growth, disease and environmental tolerance is greatly enhanced by access to a comprehensive list of gene sequences and post-genomic technologies. The current status of genomic resources is described for this species including 40 k cDNA clone collections, their associated expressed sequence tags (ESTs) and a developing series of 13 k–26 k cDNA microarrays fabricated from amplicons. The arrays have been directed at questions of response to environmental stress (cold and hypoxia), viral and bacterial disease and ectoparasite infection. Consequently, clones from a wide range of tissues were prepared. The authors discuss how these resources were generated and their application. Evidence is presented supporting that the carp microarray may also be useful as a heterologous set of probes in studies of other fish species.
We searched for sex-specific DNA sequences in the male and female genomes of African catfish, Cla... more We searched for sex-specific DNA sequences in the male and female genomes of African catfish, Clarias gariepinus (Burchell, 1822) by comparative random amplified polymorphic DNA (RAPD) assays performed on pooled DNA samples. Two sex-linked RAPD markers were identified from the male DNA pool and confirmed on individual samples, showing good agreement with phenotypic sex. Both markers were isolated, cloned and characterized. The first marker (CgaY1) was nearly 2.6 kb long, while the length of second one (CgaY2) was 458 bp. Southern blot analysis with a CgaY1 probe showed strong hybridizing fragments only in males and not in females under stringent conditions, indicating the presence of multiple copies of CgaY1 in the male genome. When tested by zoo blot on the genomes of two closely related species from the Clariidae family, CgaY1 hybridized to the DNA of Heterobranchus longifilis and generated a faint male-specific band at low stringency. CgaY2 produced similar hybridization pattern in both sexes of C. gariepinus, C. macrocephalus and H. longifilis. Specific primers were designed to the sequences and the markers were amplified in multiplex PCR reactions together with a control band common to all individuals. This allowed for rapid, molecular sexing of the species on the basis of a simple three band (male) versus one band (female) pattern. According to our knowledge these are the first sex-specific DNA markers isolated from a siluroid fish species.
We developed a simple and inexpensive method to extract DNA from fresh and preserved fish scales.... more We developed a simple and inexpensive method to extract DNA from fresh and preserved fish scales. The procedure is based on boiling the scales in 5% Chelex 100, followed by digestion with proteinase K and subsequent absorption of genomic DNA using silica. A single fresh scale from larger species (e.g., tilapia) or a few scales from smaller species (e.g., 4 scales from zebrafish) provide over 200 ng of DNA, enough for at least 40 polymerase chain reaction amplifications. The procedure is applicable for DNA isolation not only from fresh and ethanol-preserved scales, but also from dried and formaldehyde-treated samples, and thus might be useful for investigating specimens stored in museums and other collections. Since the removal of a few scales is a gentle means of sample collection, this technique will allow analysis of genetic diversity, mating systems, and parentage in populations of endangered or ornamental fish with minimal experimental influence.
We isolated novel dinucleotide, trinucleotide, and tetranucleotide microsatellites from the genom... more We isolated novel dinucleotide, trinucleotide, and tetranucleotide microsatellites from the genome of Asian sea bass (Lates calcarifer). Two genomic DNA libraries were established, one was enriched for (CA)n repeats, while the other for (GATA)n, (GACA)n, and (AAC)n repeats. Sixty clones containing an insert between 250 and 1000 bp in size were sequenced from each library; altogether 50 (43%) of them contained microsatellites. Forty microsatellites were characterized in 16 unrelated Asian sea bass individuals. Twenty-eight of them (70%) showed specific amplification and polymorphism. The allele number per loci varied between 2 and 20 with an average of 5.3, while expected heterozygosity ranged from 0.31 to 0.95 with an average of 0.64. At some loci allele sizes spread over a wide range (>100 bp). No significant correlation (r = 0.23, df = 31, P > 0.05) was found between the repeat number and the number of alleles. A whole broodstock containing 170 individuals was analyzed by using 8 selected polymorphic microsatellites. The average number of alleles per locus was 11.8 (range, 4–21). The expected heterozygosity ranged from 0.57 to 0.90 with an average of 0.75, while the fixation index was 0.02. Genetic similarity between individuals ranged from 0 to 0.72. Comparison of allele frequencies between the broodstock and the 24 nonrelated individuals revealed some unique alleles.
In contrast to Ferguson plots based on relative mobilities, Ferguson plots of proteins in polyacr... more In contrast to Ferguson plots based on relative mobilities, Ferguson plots of proteins in polyacrylamide gel electrophoresis based on their absolute mobilities were found to be linear under unusual polymerization conditions which yield relatively wide gel fibers and a low total fiber length per unit weight, but not under previously and commonly used conditions. These linear Ferguson plots in gels of 1, 3 and 5% crosslinking intersect at a single gel concentration between 1 and 2 %T (M-point). It is postulated that the measure of free mobility of the proteins is the M-point, and not the intercept of their Ferguson plots with the mobility axis as assumed previously. This postulate abolishes the well-known paradoxical interpretation of the increase with %C of the linearly extrapolated intercept of the Ferguson plot with the log(mobility) axis (designated Yo) in terms of free mobility. The postulate is also compatible with the interpretation of the points of intersection of the Ferguson plots of oligomeric series of proteins at finite gel concentrations (designated μ-points) as their common free mobilities.
Investigations on paraquat toxicity in fishes. J Nemcsok, L Orban, B Asztalos, Z Buzas, A Nemeth,... more Investigations on paraquat toxicity in fishes. J Nemcsok, L Orban, B Asztalos, Z Buzas, A Nemeth, L Boross Water International 10:22, 79-81, 1985. The effect of paraquat in different concentrations on three fish species, carp ...
Liposome-mediated gene transfer was used to introduce large DNA constructs into zygotes of Africa... more Liposome-mediated gene transfer was used to introduce large DNA constructs into zygotes of African catfish. The technique is based on the delivery of recombinant bacteriophage lambda particles (or DNA-protamine complexes) into the cytoplasm of target cells by negatively charged, large unilamellar liposomes. Dechorionated zygotes and early fish embryos were treated with the transforming liposomes. Expression of the introduced reporter genes during the first three weeks of the development of the larvae was followed by measuring the activity of corresponding enzymes. These assays have indicated very efficient DNA uptake into the embryos.
... of them have shown quite promising preliminary results in various labs: sperm electroporation... more ... of them have shown quite promising preliminary results in various labs: sperm electroporation (Muller et al., 1992; Sin et al., 1993), electroporation of fertilized eggs (Inoueetal., 1990; Buono and Linser, 1991; Powers ... In: R. Billard and J. Marcel (Editors), Aquaculture ofCyprinids. ...
We have successfully performed interspecific androgenesis between two cyprinid species. Gamma-ray... more We have successfully performed interspecific androgenesis between two cyprinid species. Gamma-ray irradiated eggs of common carp were fertilized with fresh and cryopreserved sperm of three different goldfish varieties and the haploid embryos were then heat-...
The common carp is an important fish species satisfying ornamental, food and recreational fisheri... more The common carp is an important fish species satisfying ornamental, food and recreational fisheries’ needs worldwide, but in common with other cyprinid fishes, it is particularly renowned for its environmental tolerance. Investigating the mechanistic basis of growth, disease and environmental tolerance is greatly enhanced by access to a comprehensive list of gene sequences and post-genomic technologies. The current status of genomic resources is described for this species including 40 k cDNA clone collections, their associated expressed sequence tags (ESTs) and a developing series of 13 k–26 k cDNA microarrays fabricated from amplicons. The arrays have been directed at questions of response to environmental stress (cold and hypoxia), viral and bacterial disease and ectoparasite infection. Consequently, clones from a wide range of tissues were prepared. The authors discuss how these resources were generated and their application. Evidence is presented supporting that the carp microarray may also be useful as a heterologous set of probes in studies of other fish species.
We searched for sex-specific DNA sequences in the male and female genomes of African catfish, Cla... more We searched for sex-specific DNA sequences in the male and female genomes of African catfish, Clarias gariepinus (Burchell, 1822) by comparative random amplified polymorphic DNA (RAPD) assays performed on pooled DNA samples. Two sex-linked RAPD markers were identified from the male DNA pool and confirmed on individual samples, showing good agreement with phenotypic sex. Both markers were isolated, cloned and characterized. The first marker (CgaY1) was nearly 2.6 kb long, while the length of second one (CgaY2) was 458 bp. Southern blot analysis with a CgaY1 probe showed strong hybridizing fragments only in males and not in females under stringent conditions, indicating the presence of multiple copies of CgaY1 in the male genome. When tested by zoo blot on the genomes of two closely related species from the Clariidae family, CgaY1 hybridized to the DNA of Heterobranchus longifilis and generated a faint male-specific band at low stringency. CgaY2 produced similar hybridization pattern in both sexes of C. gariepinus, C. macrocephalus and H. longifilis. Specific primers were designed to the sequences and the markers were amplified in multiplex PCR reactions together with a control band common to all individuals. This allowed for rapid, molecular sexing of the species on the basis of a simple three band (male) versus one band (female) pattern. According to our knowledge these are the first sex-specific DNA markers isolated from a siluroid fish species.
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