Identification of genome organization in the unusual allotetraploid form of Carassius auratus gibelio

The unusual allotetraploid form with unequal contribution of chromosome sets was discovered from the gynogenetic offspring of Carassius auratus gibelio stimulated by red common carp sperm. In this study, genomic in situ hybridization (GISH) and fluorescent in situ hybridization (FISH) with 45S rDNA probe are used. The GISH results lead to the identification of species-specific chromosomes, which permits to demonstrate the origin and genome organization in the allotetraploid form. Moreover, chromosome localization of 45S rDNA and co-localizations of 45S rDNA and Cyprinus carpio genomic DNA further confirm that one extra 45S rDNA positive chromosome in the allotetraploid form originates from the paternal haploid genome of C carpio, and other 5 45S rDNA-containing chromosomes are from the maternal genome of Carassius auratus gibelio. And, the correlation between 45 rDNA and the nucleolar organizer regions (NORs) is confirmed by silver nitrate staining. The data provide direct experiment evidence that the allotetraploid actually contains three chromosome sets of Carassius auratus gibelio and one chromosome set of C carpio, and will be a useful genetic material for both basic research and breeding practice. (c) 2006 Elsevier B.V. All rights reserved.

Mutations stabilize small subunit ribosomal RNA in desiccation-tolerant cyanobacteria Nostoc

The ribosomal RNA molecule is an ideal model for evaluating the stability of a gene product under desiccation stress. We isolated 8 Nostoc strains that had the capacity to withstand desiccation in habitats and sequenced their 16S rRNA genes. The stabilities of 16S rRNAs secondary structures, indicated by free energy change of folding, were compared among Nostoc and other related species. The results suggested that 163 rRNA secondary structures of the desiccation-tolerant Nostoc strains were more stable than that of planktonic Nostocaceae species. The stabilizing mutations were divided into two categories: (1) those causing GC to replace other types of base pairs in stems and (2) those causing extension of stems. By mapping stabilizing mutations onto the Nostoc phylogenetic tree based on 16S rRNA gene, it was shown that most of stabilizing mutations had evolved during adaptive radiation among Nostoc spp. The evolution of 16S rRNA along the Nostoc lineage is suggested to be selectively advantageous under desiccation stress.

Genetic diversity: Geographical distribution and toxin profiles of Microcystis strains (Cyanobacteria) in China

Twenty strains of Microcystis Kutz were isolated from different freshwater bodies in China to analyze the diversity, geographical distribution and toxin profiles. Based on whole-cell polymerase chain reaction of cpcBA-IGS nucleotide sequence, the derived neighbor-joining (NJ) and maximum parsimony (MP) trees indicate that these strains of Microcystis can be divided into four clusters. The strains from south, middle and north region of China formed distinct lineages, suggesting high diversity and a geographical distribution from south to north locations. Moreover, the results being indicating high variable genotypes of the strains of the Microcystis strains from the same lake show that there is high diversity of Microcystis within a water bloom population. Comparing the results of the present study with those reported for compared with 43 strains of Microcystis from other locations, also reveals Chinese strains have high similarity with those from regions in the North Hemispherical. This suggests that the Microcystis strains in the world might have a geographical distribution. Analysis of 30 strains using the primers MCF/TER and TOX2P/TOX2M showed that there was no correlation between the gene of cpcBA-IGS and the presence of mcy. Toxic strains were founded to be predominant in different water bodies throughout China.

Effect of dietary cyanobacteria on growth and accumulation of microcystins in Nile tilapia (Oreochromis niloticus)

A 12-week growth trial was conducted in a flow-through system to investigate the chronic toxic effect of dietary intake of cyanobacteria on growth, feed utilization and microcystins accumulation in Nile tilapia (Oreochromis niloticus L.) (initial body weight: 5.6 g). Six isonitrogenous and isocaloric diets were formulated to include different contents of cyanobacteria with the dietary microcystins increasing from 0 to 5460.06 ng/g diet. The results showed that dietary intake of cyanobacteria could increase the growth of tilapia while there are no impacts on feed conversion efficiency or mortality. Feeding rate was higher for the diets containing highest cyanobacteria. Microcystins were mostly accumulated in fish liver. The relationship between microcystins contents in muscle, liver, spleen and dietary intake could be described by quadratic equations. Microcystins content in the muscle of Nile tilapia in present study exceeded the upper limit of the tolerable daily intake (TDI) of microcystins suggested by the WHO (0.04 mu g/kg body weight/d). It is suggested that Nile tilapia fed on toxic cyanobacteria is not suitable for human food. (c) 2006 Elsevier B.V. All rights reserved.

Sox genes in grass carp (Ctenopharyngodon idella) with their implications for genome duplication and evolution

The Sox gene family is found in a broad range of animal taxa and encodes important gene regulatory proteins involved in a variety of developmental processes. We have obtained clones representing the HMG boxes of twelve Sox genes from grass carp (Ctenopharyngodon idella), one of the four major domestic carps in China. The cloned Sox genes belong to group B1, B2 and C. Our analyses show that whereas the human genome contains a single copy of Sox4, Sox11 and Sox14, each of these genes has two co-orthologs in grass carp, and the duplication of Sox4 and Sox11 occurred before the divergence of grass carp and zebrafish, which support the "fish-specific whole-genome duplication" theory. An estimation for the origin of grass carp based on the molecular clock using Sox1, Sox3 and Sox11 genes as markers indicates that grass carp (subfamily Leuciscinae) and zebrafish (subfamily Danioninae) diverged approximately 60 million years ago. The potential uses of Sox genes as markers in revealing the evolutionary history of grass carp are discussed.

The complete mitochondrial genome of the helmet catfish Cranoglanis bouderius (Silurifonnes : Cranoglanididae) and the phylogeny of otophysan fishes

The complete sequence of the 16,539 nucleotide mitochondrial genome from the single species of the catfish family Cranoglanididae, the helmet catfish Cranoglanis bouderius, was determined using the long and accurate polymerase chain reaction (LA PCR) method. The nucleotide sequences of C. bouderius mitochondrial DNA have been compared with those of three other catfish species in the same order. The contents of the C. bouderius mitochondrial genome are 13 protein-coding genes, two ribosomal RNA and 22 transfer RNA genes, and a non-coding control region, the gene order of which is identical to that observed in most other vertebrates. Phylogenetic analyses for 13 otophysan fishes were performed using Bayesian method based on the concatenated mtDNA protein-coding gene sequence and the individual protein-coding gene sequence data set. The competing otophysan topologies were then tested by using the approximately unbiased test, the Kishino-Hasegawa test, and the Shimodaira-Hasegawa test. The results show that the grouping ((((Characifonnes, Gymnotiformes), Siluriformes), Cyprinifionnes), outgroup) is the most likely but there is no significant difference between this one and the other alternative hypotheses. In addition, the phylogenetic placement of the family Cranoglanididae among siluriform families was also discussed. (c) 2006 Elsevier B.V. All rights reserved.

The functional divergence of two glgP homologues in Synechocystis sp PCC 6803

Glycogen phosphorylase (GlgP, EC 2.4.1.1) catalyzes the cleavage of glycogen into glucose-1-phosphate (Glc-1-P), the first step in glycogen catabolism. Two glgP homologues are found in the genome of Synechocystis sp. PCC 6803, a unicellular cyanobacterium: sll1356 and slr1367. We report on the different functions of these glgP homologues. sll1356, rather than slr1367, is essential for growth at high temperatures. On the other hand, when CO2-fixation and the supply of glucose are both limited, slr1367 is the key factor in glycogen metabolism. In cells growing autotrophically, sll1356 plays a more important role in glycogen digestion than slr1367. This functional divergence is also supported by a phylogenetic analysis of glgP homologues in cyanobacteria.

Growth and antioxidant system of the cyanobacterium Synechococcus elongatus in response to microcystin-RR

Microcystins, one type of the cyanobacterial toxins, show a broad range of hazardous effects on other organisms. Most of the researches on the toxic effects of microcystins have involved in animals and higher plants. Little work, however, has been done on evaluating the mechanisms of microcystin toxicity on algae. In this study, the toxicological effects of microcystin-RR (MC-RR) on the cyanobacterium Synechococcus elongatus were investigated. For this purpose, six physio-biochemical parameters (cell optical density, reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH), glutathione peroxidase (GSH-Px) and glutathione S-transferase (GST)) were tested in algal cells when exposed to 100 mug(-1) microcystin-RR. The results showed that the growth of Synechococcus elongatus ( expressed as optical density) was significantly inhibited compared with the control. At the same time, the treated algae exhibited a pronounced increase in production of ROS and MDA after 6 days exposure to microcystin-RR. Signi. cant changes in GSH levels and GSH-Px, GSH activities were also detected in algal cells, with higher values being observed in the toxin treated algae after 6 days exposure. GST activities in the treated algae exhibited a decline after exposure and rapid augmentation on day 3, thereafter, they kept at a high level when compared to the control group. GSH contents and GSH-Px activities were also significantly raised in the toxin-treated algae cells from day 3, but they showed a sharp decrease on day 4, which was the onward of cell proliferation. These results suggested that oxidative stress manifested by elevated ROS levels and MDA contents might be responsible for the toxicity of microcystin to Synechococcus elongatus and the algal cells could improve their antioxidant ability through the enhancement of enzymatic and non-enzymatic preventive substances.

Complete genome sequence of lymphocystis disease virus isolated from China

Lymphocystis diseases in fish throughout the world have been extensively described. Here we report the complete genome sequence of lymphocystis disease virus isolated in China (LCDV-C), an LCDV isolated from cultured flounder (Paralichthys olivaceus) with lymphocystis disease in China. The LCDV-C genome is 186,250 bp, with a base composition of 27.25% G+C. Computer-assisted analysis revealed 240 potential open reading frames (ORFs) and 176 nonoverlapping putative viral genes, which encode polypeptides ranging from 40 to 1,193 amino acids. The percent coding density is 67%, and the average length of each ORF is 702 bp. A search of the GenBank database using the 176 individual putative genes revealed 103 homologues to the corresponding ORFs of LCDV-1 and 73 potential genes that were not found in LCDV-1 and other iridoviruses. Among the 73 genes, there are 8 genes that contain conserved domains of cellular genes and 65 novel genes that do not show any significant homology with the sequences in public databases. Although a certain extent of similarity between putative gene products of LCDV-C and corresponding proteins of LCDV-1 was revealed, no colinearity was detected when their ORF arrangements and coding strategies were compared to each other, suggesting that a high degree of genetic rearrangements between them has occurred. And a large number of tandem and overlapping repeated sequences were observed in the LCDV-C genome. The deduced amino acid sequence of the major capsid protein (MCP) presents the highest identity to those of LCDV-1 and other iridoviruses among the LCDV-C gene products. Furthermore, a phylogenetic tree was constructed based on the multiple alignments of nine MCP amino acid sequences. Interestingly, LCDV-C and LCDV-1 were clustered together, but their amino acid identity is much less than that in other clusters. The unexpected levels of divergence between their genomes in size, gene organization, and gene product identity suggest that LCDV-C and LCDV-1 shouldn't belong to a same species and that LCDV-C should be considered a species different from LCDV-1.

Cloning of biologically active genomes from a Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus isolate by using a bacterial artificial chromosome

Purification of genotypes from baculovirus isolates provides understanding of the diversity of baculoviruses and may lead to the development of better pesticides. Here, we report the cloning of different genotypes from an isolate of Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV) by using a bacterial artificial chromosome (BAC). A transfer vector (pHZB10) was constructed which contained an Escherichia coli mini-F replicon cassette within the upstream and downstream arms of HaSNPV polyhedrin gene. Hz2e5 cells were co-transfected with wild-type HaSNPV DNA and pHZB10 to generate recombinant viruses by homologous recombination. The DNA of budded viruses (BVs) was used to transform E. coli. One of the bacmid colonies, HaBacHZ8, has restriction enzyme digestion profiles similar to an in vivo cloned strain HaSNPV-G4, the genome of which has been completely sequenced. For testing the oral infectivity, the polyhedrin gene of HaSNPV was reintroduced into HaBacHZ8 to generate the recombinant bacmid HaBacDF6. The results of one-step growth curves, electron microscopic examination, protein expression analysis and bioassays indicated that HaBacDF6 replicated as well as HaSNPV-G4 in vitro and in vivo. The biologically functional HaSNPV bacmids obtained in this research will facilitate future studies on the function genomics and genetic modification of HaSNPV. (C) 2003 Elsevier B.V. All rights reserved.

Two unisexual artificial polyploid clones constructed by genome addition of common carp (Cyprinus carp) and crucian carp (Carassius auratus)

A polyploid hybrid fish with natural gynogenesis can prevent segregation and maintain their hybrid vigor in their progenies. Supposing the reproduction mode of induced polyploid fish being natural gynogenesis, allopolyploid hybrid between common carp and crucian carp into allopolyploid was performed. The purpose of this paper is to describe a lineage from sexual diploid carp transforming into allotriploid and allotetraploid unisexual clones by genome addition. The diploid hybrid between common carp and crucian carp reproduces an unreduced nucleus consisting of two parental genomes. This unreduced female pronucleus will fuse with male pronucleus and form allotriploid zygote after penetration of related species sperms. Allotriploid embryos grow normally, and part of female allotriploid can produce unreduced mature ova with three genomes. Mature ova of most allotriploid females are provided with natural gynogenetic trait and their nuclei do not fuse with any entrance sperm. All female offspring are produced by gynogenesis of allotriploid egg under activation of penetrating sperms. These offspring maintain morphological traits of their allotriploid maternal and form an allotetraploid unisexual clone by gynogenetic reproduction mode. However, female nuclei of rare allotriploid female can fuse with penetrating male pronuclei and result in the appearance of allotetraploid individuals by means of genome addition. All allotetraploid females can reproduce unreduced mature eggs containing four genomes. Therefore, mature eggs of allotetraploid maintain gynogenetic trait and allotetraploid unisexual clone is produced under activation of related species sperms.

The vertical microdistribution of cyanobacteria and green algae within desert crusts and the development of the algal crusts

Substantial amounts of algal crusts were collected from five different desert experimental sites aged 42, 34, 17, 8 and 4 years, respectively, at Shapotou ( China) and analyzed at a 0.1 mm microscale of depth. It was found that the vertical distribution of cyanobacteria and microalgae in the crusts was distinctly laminated into an inorganic-layer (ca. 0.00 - 0.02 mm, with few algae), an algae-dense-layer ( ca. 0.02 - 1.0 mm) and an algae-sparse-layer ( ca. 1.0 - 5.0 mm). It was interesting to note that in all crusts Scytonema javanicum Born et Flah ( or Nostoc sp., cyanobacterium), Desmococcus olivaceus (Pers ex Ach., green alga) Laundon and Microcoleus vaginatus Gom. ( cyanobacterium) dominated at the depth of 0.02 - 0.05, 0.05 - 0.1 and 0.1 - 1.0 mm, respectively, from the surface. Phormidium tenue Gom. ( or Lyngbya cryptovaginatus Schk., cyanobacterium) and Navicula cryptocephala Kutz.( or Hantzschia amphioxys (Ehr.) Grun. and N. cryptocephala together, diatom) dominated at the depth of 1.0 - 3.0 and 3.5 - 4.0 mm, respectively, of the crusts from the 42 and 34 year old sites. It was apparent that in more developed crusts there were more green algae and the niches of Nostoc sp., Chlorella vulgaris Beij., M. vaginatus, N. cryptocephala and fungi were nearer to the surface. If lichens and mosses accounted for less than 41.5% of the crust surface, algal biovolume was bigger when the crust was older, but the opposite was true when the cryptogams other than algae covered more than 70%. In addition to detailed species composition and biovolume, analyses of soil physicochemical properties, micromorphologies and mineral components were also performed. It was found that the concentration of organic matter and nutrients, electric conductivity, silt, clay, secondary minerals were higher and there were more micro-beddings in the older crusts than the less developed ones. Possible mechanisms for the algal vertical microdistribtion at different stages and the impact of soil topography on crust development are discussed. It is concluded that biomethods ( such as fine species distribution and biovolume) were more precise than mineralogical approaches in judging algal crust development and thus could be a better means to measure the potentiality of algal crusts in desert amelioration.

A TPR-family membrane protein gene is required for light-activated heterotrophic growth of the cyanobacterium Synechocystis sp PCC 6803

The unicellular cyanobacterium Synechocystis sp. PCC6803 can grow heterotrophically in complete darkness, given that a brief period of illumination is supplemented every day (light-activated heterotrophic growth, LAHG), or under very weak ( < 0.5 mumol m(-2) s(-1)) but continuous light. By random insertion of the genome with an antibiotic resistance cassette, mutants defective in LAHG were generated. In two identical mutants, sll0886, a tetratricopeptide repeat (TPR)-family membrane protein gene, was disrupted. Targeted insertion of sll0886 and three downstream genes showed that the phenotype was not due to a polar effect. The sll0886 mutant shows normal photoheterotrophic growth when the light intensity is at 2.5 mumol m(-2) s(-1) or above, but no growth at 0.5 mumol m(-2) s(-1). Homologs to sll0886 are also present in cyanobacteria that are not known of LAHG. sll0886 and homologs may be involved in controlling different physiological processes that respond to light of low fluence. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Taxonomic revision of water-bloom-forming species of oscillatorioid cyanobacteria

A polyphasic approach was used to clarify the taxonomy of the water-bloom-forming oscillatorioid cyanobacteria. Seventy-five strains of oscillatorioid cyanobacteria were characterized by 16S rDNA sequence analysis, DNA base composition, DNA-DNA hybridization, fatty acid composition, phycobilin pigment composition, complementary chromatic adaptation, morphological characters, growth temperature and salinity tolerance. Phylogenetic analysis based on 165 rDNA sequences divided the strains into six groups, all of which were clearly separated from the type species of the genus Oscillatoria, Oscillatoria princeps Gomont NIVA CYA 150. Therefore, these strains should be classified into genera other than Oscillatoria. Groups I-III were closely related to one another and groups IV-VI were distinct from one another and from groups I to III. Group I was further divided into two subgroups, group I-pc, which includes strains containing only phycocyanin (PC), and group I-pe, which includes strains containing large amounts of phycoerythrin (PE) in addition to PC. This phenotypic distinction was supported by DNA-DNA hybridization studies. Based on the properties examined herein and data from traditional, botanical taxonomic studies, the groups and subgroups were classified into single species and we propose either emended or new taxonomic descriptions for Planktothrix agardhii (type strain NIES 204(T)), Planktothrix rubescens (type strain CCAP 1459/22(T)) Planktothrix pseudagardhii sp. nov. (type strain T1-8-4(T)), Planktothrix mougeotii (type strain TR1-5(T)), Planktothricoides raciborskii gen. nov., comb. nov. (type strain NIES 207(T)), Tychonema bourrellyi (type strain CCAP 1459/11B(T)) and Limnothrix redekei (type strain NIVA CYA 277/1(T)).

Common evolutionary origin of aquareoviruses and orthoreoviruses revealed by genome characterization of Golden shiner reovirus, Grass carp reovirus, Striped bass reovirus and golden ide reovirus (genus Aquareovirus, family Reoviridae)

Full-length and partial genome sequences of four members of the genus Aquareovirus, family Reoviridae (Golden shiner reovirus, Grass carp reovirus, Striped bass reovirus and golden ide reovirus) were characterized. Based on sequence comparison, the unclassified Grass carp reovirus was shown to be a member of the species Aquareovirus C The status of golden ide reovirus, another unclassified aquareovirus, was also examined. Sequence analysis showed that it did not belong to the species Aquareovirus A or C, but assessment of its relationship to the species Aquareovirus B, D, E and F was hampered by the absence of genetic data from these species. In agreement with previous reports of ultrastructural resemblance between aquareoviruses and orthoreoviruses, genetic analysis revealed homology in the genes of the two groups. This homology concerned eight of the 11 segments of the aquareovirus genome (amino acid identity 17-42%), and similar genetic organization was observed in two other segments. The conserved terminal sequences in the genomes of members of the two groups were also similar. These data are undoubtedly an indication of the common evolutionary origin of these viruses. This clear genetic relatedness between members of distinct genera is unique within the family Reoviridae. Such a genetic relationship is usually observed between members of a single genus. However, the current taxonomic classification of aquareoviruses and orthoreoviruses in two different genera is supported by a number of characteristics, including their distinct G+C contents, unequal numbers of genome segments, absence of an antigenic relationship, different cytopathic effects and specific econiches.