The complete nucleotide sequence and gene organization of carp (Cyprinus carpio) mitochondrial genome

The complete sequence of the carp mitochondrial genome of 16,575 base pairs has been determined. The carp mitochondrial genome encodes the same set of genes (13 proteins, 2 rRNAs, and 22 tRNAs) as do other vertebrate mitochondrial DNAs. Comparison of this teleostean mitochondrial genome with those of other vertebrates reveals a similar gene order and compact genomic organization. The codon usage of proteins of carp mitochondrial genome is similar to that of other vertebrates. The phylogenetic relationship for mitochondrial protein genes is more apparent than that for the mitochondrial tRNA and rRNA genes.Correspondence to: F. Huang     

Taenia hydatigena: isolation of mitochondrial DNA, molecular cloning, and physical mitochondrial genome mapping

Mitochondrial DNA was isolated from Taenia hydatigena, T. crassiceps, and Echinococcus granulosus using a cetyltrimethylammonium bromide precipitation technique. The technique is simple, rapid, reproducible, and does not require extensive high speed ultracentrifugation. The advantage of using mitochondrial DNA from taeniid cestodes for comparative restriction analysis was demonstrated. Mitochondrial DNA of T. hydatigena was isolated as covalently closed circular molecules. These were linearized by single digestion with BamHI and the molecular weight was estimated from the linear form of 17.6 kb. The mitochondrial DNA of T. hydatigena is therefore similar in size and structure to that of many other animal species. The entire mitochondrial genome was cloned into pBR322 in Escherichia coli and a restriction map of the recombinant molecule was constructed. The potential of using the cloned mitochondrial genome as a probe in speciation studies as well as for providing functional information on the role of the cestode mitochondrion is discussed.     

Genetic divergence between Cyprinus carpio carpio and Cyprinus carpio haematopterus as assessed by mitochondrial DNA analysis,with emphasis on origin of European domestic carp

Although common carp is the major fish species in Asian and European aquaculture and many domestic varieties have occurred, there is a controversy about the origination of European domestic common carp. Some scientists affirmed that the ancestor of European domestic common carp was Danube River wild common carp, but others considered it might be Asian common carp. For elucidating origination of European domestic common carp, we chose two representative European domestic common carp strains (German mirror carp and Russian scattered scaled mirror carp) and one wild common carp strain of Cyprinus carpio carpio subspecies (Volga River wild common carp) and two Asian common carp strains, the Yangtze River wild common carp (Cyprinus carpio haematopterus) and traditionally domestic Xingguo red common carp, as experimental materials. ND5–ND6 and D-loop segments of mitochondrial DNA were amplified by polymerase chain reaction and analyzed through restriction fragment length polymorphism (RFLP) and sequencing respectively. The results revealed that HaeIII and DdeI digestion patterns of ND5–ND6 segment and sequences of control region were different between European subspecies C. carpio carpio and Asian subspecies C. carpio haematopterus. Phylogenetic analysis showed that German mirror carp and Russian scattered scaled mirror carp belonged to two subspecies, C. carpio carpio and C. carpio haematopterus, respectively. Therefore, there were different ancestors for domestic carp in Europe: German mirror carp was domesticated from European subspecies C. carpio carpio and Russian scattered scaled mirror carp originated from Asian subspecies C. carpio haematopterus.     

Tripartite mitochondrial genome of spinach: physical structure, mitochondrial gene mapping, and locations of transposed chloroplast DNA sequences.

A complete physical map of the spinach mitochondrial genome has been established. The entire sequence content of 327 kilobase pairs (kb) is postulated to occur as a single circular molecule. Two directly repeated elements of approximately 6 kb, located on this "master chromosome", are proposed to participate in an intragenomic recombination event that reversibly generates two "subgenomic" circles of 93 kb and 234 kb. The positions of protein and ribosomal RNA-encoding genes, determined by heterologous filter hybridizations, are scattered throughout the genome, with duplicate 26S rRNA genes located partially or entirely within the 6 kb repeat elements. Filter hybridizations between spinach mitochondrial DNA and cloned segments of spinach chloroplast DNA reveal at least twelve dispersed regions of inter-organellar sequence homology.     

鲤鱼mtDNA酶切图谱的构建

采用密度梯度离心法及ＲＮａｓｅ消化法制备并纯化了鲤（ＧｙｐｒｉｎｕｓｃａｒｐｉｏＬｉｎｎａｅｕｓ）肝脏线粒体ＤＮＡ（ｍｔＤＮＡ）,用１０种限制性内切酶对ｍｔＤＮＡ进行了分析,鲤鱼ｍｔＤＮＡ分子量约１０．１２×１０￣６,约１６．４９ｋｂ．ＳａｌⅠ、ＰｓｔⅠ、ＢａｍＨⅠ、ＸｂａⅠ、ＢｇｌⅠ、ＰｖｕⅡ、ＸｈｏⅠ、ＥｃｏＲⅠ、ＤｒａⅠ和ＨｉｎｄⅢ分别为１、１、３、３、３、４、１、４、４、和６个切点。根据单酶解及双酶解结果,构建了鲤ｍｔＤＮＡ１０种具酶３０个切点的限制性酶切图谱。     

Cloning and physical mapping of Herpesvirus sylvilagus DNA

The physical map positions for the EcoRI restriction fragments of Herpesvirus sylvilagus DNA were determined using cloned virus DNA fragments as probes for hybridization. Approximately 75% of the virus genome was cloned. The internal location of the repeated nucleotide sequences and the lack of isomeric forms suggested that the virus genome belongs to the class C herpesvirus DNA.     

Characterization of the 42-S nucleoprotein particles of Cyprinus carpio oocytes

Previtellogenic oocytes of the fish Cyprinus carpio contain 42S nucleoprotein particles that are composed of two proteins of molecular weights 48,500 and 39,300 (molar ratio 2:1), tRNA and 5S RNA (molar ratio 3:1). The tRNA population embodied in the 42S particle contains all amino acid acceptor species but their distribution differs from that found in tRNA from mature oocytes.     

Cloning, characterization and expression analysis of interleukin-10 from the common carp, Cyprinus carpio L.

Interleukin (IL)-10 was cloned from the common carp (Cyprinus carpio L.) using IL-10 primers from carp head kidney following stimulation with concanavalin A and lipopolysaccharide. The cDNA consisted of a 1096 bp sequence containing a 55 bp 5' untranslated region and a 498 bp 3' untranslated region. An open reading frame of 543 bp encoded a putative 180 amino acid protein with a putative signal peptide of 22 amino acids. The signature motif of IL-10 is conserved in carp sequence. A 2083 bp genomic sequence of carp IL-10 was found to contain five exons interrupted by four introns. With the exception of much more compact introns, the genomic structure was similar to that of mammalian IL-10. By homology, phylogeny and genomic analyses, the carp gene cloned was designated as IL-10. Carp IL-10 was expressed in head, kidney, liver, spleen and intestine during the resting phase. The gene was also expressed in head kidney and liver following in vitro stimulation with lipopolysaccharide.     

The mitochondrial DNA of Dictyostelium discoideum: complete sequence, gene content and genome organization

We present an overview of the gene content and organization of the mitochondrial genome of Dictyostelium discoideum. The mitochondria genome consists of 55,564 bp with an A + T content of 72.6%. The identified genes include those for two ribosomal RNAs (rnl and rns), 18 tRNAs, ten subunits of the NADH dehydrogenase complex (nad1, 2, 3, 4, 4L, 5, 6, 7, 9 and 11), apocytochrome b (cytb), three subunits of the cytochrome oxidase (cox1/2 and 3), four subunits of the ATP synthase complex (atp1, 6, 8 and 9), 15 ribosomal proteins, and five other ORFs, excluding intronic ORFs. Notable features of D. discoideum mtDNA include the following. (1) All genes are encoded on the same strand of the DNA and a universal genetic code is used. (2) The cox1 gene has no termination codon and is fused to the downstream cox2 gene. The 13 genes for ribosomal proteins and four ORF genes form a cluster 15.4 kb long with several gene overlaps. (3) The number of tRNAs encoded in the genome is not sufficient to support the synthesis of mitochondrial protein. (4) In total, five group I introns reside in rnl and cox1/2, and three of those in cox1/2 contain four free-standing ORFs. We compare the genome to other sequenced mitochondrial genomes, particularly that of Acanthamoeba castellanii. Received: 5 July 1999 / Accepted: 17 January 2000     

Structure,organization and expression of common carp (Cyprinus carpio L.) NKEF-B gene

Natural killer (NK) cell enhancing factor (NKEF) belongs to the newly defined peroxiredoxin (Prx) family. Its functions are to enhance NK cell cytotoxicity and to protect DNA and proteins from oxidative damage. In this study, a partial cDNA sequence of carp NKEF-B was isolated from thymus cDNA library. Subsequently, the full-length cDNA of carp NKEF-B was obtained by means of 3′ and 5′ RACE, respectively. The full-length cDNA of carp NKEF-B was 1022 bp, consisting of a 73 bp 5′-terminal untranslated region (UTR), a 355 bp 3′-terminal UTR, and a 594 bp open reading frame coding for a protein of 197 amino acids. Carp NKEF-B contained two consensus Val-Cys-Pro (VCP) motifs and three consensus cysteine (Cys-51, Cys-70 and Cys-172) residues. Sequence comparison showed that the deduced amino acid sequence of carp NKEF-B had an overall similarity of 74–96% to that of other species homologues. Phylogenetic analysis revealed that carp NKEF-B forms a cluster with other known teleost NKEF-Bs. Then, by PCR we obtained a 5.1-k long genomic DNA of carp NKEF-B containing six exons and five introns. Real-time RT-PCR results showed that carp NKEF-B gene was predominantly detected in kidney and head kidney under un-infected conditions. Whereas under SVCV-infection condition, the expression of NKEF-B gene was significantly increased in blood cells, gill, intestine and spleen, but maintained in liver, and decreased significantly in kidney and head kidney. Finally, the rNKEF-B was constructed and expressed in Escherichia coli. By using an antibody against carp rNKEF-B, immunohistochemical study further indicated that NKEF-B positive cells are mainly some RBCs and a few epithelial cells in gill and intestine, and that under SVCV-infection condition, these positive cells or positive products in their cytoplasm were mainly increased in gill and spleen sections of carp. The results obtained in the present study will help to understand the function of NKEF-B in teleost innate immunity.     

Evolution of mammalian genome organization inferred from comparative gene mapping

Comparative genome analyses, including chromosome painting in over 40 diverse mammalian species, ordered gene maps from several representatives of different mammalian and vertebrate orders, and large-scale sequencing of the human and mouse genomes are beginning to provide insight into the rates and patterns of chromosomal evolution on a whole-genome scale, as well as into the forces that have sculpted the genomes of extant mammalian species.     

Genetic diversity and population structure inferred from the partially duplicated genome of domesticated carp,Cyprinus carpio L.

Genetic relationships among eight populations of domesticated carp (Cyprinus carpio L.), a species with a partially duplicated genome, were studied using 12 microsatellites and 505 AFLP bands. The populations included three aquacultured carp strains and five ornamental carp (koi) variants. Grass carp (Ctenopharyngodon idella) was used as an outgroup. AFLP-based gene diversity varied from 5% (grass carp) to 32% (koi) and reflected the reasonably well understood histories and breeding practices of the populations. A large fraction of the molecular variance was due to differences between aquacultured and ornamental carps. Further analyses based on microsatellite data, including cluster analysis and neighbor-joining trees, supported the genetic distinctiveness of aquacultured and ornamental carps, despite the recent divergence of the two groups. In contrast to what was observed for AFLP-based diversity, the frequency of heterozygotes based on microsatellites was comparable among all populations. This discrepancy can potentially be explained by duplication of some loci in Cyprinus carpio L., and a model that shows how duplication can increase heterozygosity estimates for microsatellites but not for AFLP loci is discussed. Our analyses in carp can help in understanding the consequences of genotyping duplicated loci and in interpreting discrepancies between dominant and co-dominant markers in species with recent genome duplication.     

Genetic association mapping and genome organization of maize

Association mapping, a high-resolution method for mapping quantitative trait loci based on linkage disequilibrium, holds great promise for the dissection of complex genetic traits. The recent assembly and characterization of maize association mapping panels, development of improved statistical methods, and successful association of candidate genes have begun to realize the power of candidate-gene association mapping. Although the complexity of the maize genome poses several significant challenges to the application of association mapping, the ongoing genome sequencing project will ultimately allow for a thorough genome-wide examination of nucleotide polymorphism-trait association.     

The mitochondrial genome of the firefly, Pyrocoelia rufa: complete DNA sequence, genome organization, and phylogenetic analysis with other insects

The complete nucleotide sequences of the mt genome from the firefly, Pyrococelia rufa (Coeleoptera: Lampyridae) was determined. The circular genome is 17,739-bp long, and contains a typical gene complement, order, and arrangement identical to Drosophila yacuba. The presence of 1,724-bp long intergenic spacer in the P. rufa mt genome is unique. The putative initiation codon for ND1 gene appears to be TTG, instead of frequently found ATN. All tRNAs showed stable canonical clover-leaf structure of other mt tRNAs, except for tRNA(Ser) (AGN), DHU arm of which could not form stable stem-loop structure. Phylogenetic analysis among insect orders confirmed a monophyletic Endopterygota, a monophyletic Mecopterida, a monophyletic Diptera, a monophyletic Lepidoptera, and a monophyletic Coleoptera, suggesting that the complete insect mt genome sequence has a resolving power in the diversification events within Endopterygota. However, internal relationships among three coleopteran species are not clear, and the inclusion of some insect orders (i.e., apterygotan T. gertschi) in the analysis provided inconsistent results compared to other molecular studies.     

Description of an as yet unclassified DNA virus from diseased Cyprinus carpio species

Numerous deaths of koi and common carp (Cyprinus carpio) were observed on many farms throughout Israel, resulting in severe financial losses. The lethal viral disease observed is highly contagious and extremely virulent, but morbidity and mortality are restricted to koi and common carp populations. Diseased fish exhibit fatigue and gasping movements in shallow water. Infected fish had interstitial nephritis and gill necrosis as well as petechial hemorrhages in the liver and other symptoms that were not consistent with viral disease, suggesting a secondary infection. Here we report the isolation of carp nephritis and gill necrosis virus (CNGV), which is the etiologic agent of this disease. The virus propagates and induces severe cytopathic effects by 5 days postinfection in fresh koi or carp fin cell cultures (KFC and CFC, respectively), but not in epithelioma papillosum cyprini cells. The virus harvested from KFC cultures induced the same clinical signs, with a mortality of 75 to 95%, upon inoculation into naive koi and common carp. Using PCR, we provide final proof that the isolated virus is indeed the etiologic agent of food and ornamental carp mortalities in fish husbandry. Electron microscopy revealed viral cores with icosahedral morphology of 100 to 110 nm that resembled herpesviruses. Electron micrographs of purified pelleted CNGV sections, together with viral sensitivities to ether and Triton X-100, suggested that it is an enveloped virus. However, the genome of the isolated virus is a double-stranded DNA (dsDNA) molecule of 270 to 290 kbp, which is larger than known herpesviruses. The viral DNA seems highly divergent and bears only small fragments (16 to 45 bp) that are similar to the genomes of several DNA viruses. Nevertheless, amino acid sequences encoded by CNGV DNA fragments bear similarities primarily to members of the Poxviridae and Herpesviridae and to other large dsDNA viruses. We suggest, therefore, that the etiologic agent of this disease may represent an as yet unclassified virus species that is endemic in C. carpio (carp).     

The organization and inheritance of the mitochondrial genome

Mitochondrial DNA (mtDNA) encodes essential components of the cellular energy-producing apparatus, and lesions in mtDNA and mitochondrial dysfunction contribute to numerous human diseases. Understanding mtDNA organization and inheritance is therefore an important goal. Recent studies have revealed that mitochondria use diverse metabolic enzymes to organize and protect mtDNA, drive the segregation of the organellar genome, and couple the inheritance of mtDNA with cellular metabolism. In addition, components of a membrane-associated mtDNA segregation apparatus that might link mtDNA transmission to mitochondrial movements are beginning to be identified. These findings provide new insights into the mechanisms of mtDNA maintenance and inheritance.