Molecular cloning of the common carp (Cyprinus carpio) rhodopsin cDNA

A recombinant phage clone containing a 1584 nucleotides rhodopsin cDNA was screened from a carp retinal cDNA library. The inserted DNA consisting of a single open reading frame of 1062 nucleotides at positions 72 to 1133 encodes a 354 amino acid polypeptide. The deduced amino acid sequence of carp rhodopsin showed 95.7, 85.5 and 74.4% identity with that of goldfish, sand goby and lamprey, respectively. The sites of palmitoylation, glycosylation, disulfide bond formation and Schiff base formation in the putative rhodopsin are all conserved.     

Microsatellite markers in common carp (Cyprinus carpio L.)

Microsatellite markers of the poly (CA) type in common carp ( Cyprinus carpio L.) are described. Clones containing a (CA) repeat were isolated from a common carp genomic library and sequenced. The number of repeats found was high compared to mammals but comparable with other teleost fishes. Classification of the repeats (perfect, imperfect and compound) are compared with the Atlantic cod ( Gadus morhua L.), rainbow trout ( Oncorhynchus mykiss ), and Atlantic salmon ( Salmo salar L.). A total of 41 primer sets were designed and tested for polymorphism on a test panel of eight animals (derived from outbred lines, inbred lines and gynogenetic clones). Thirty-two markers were found to be polymorphic. The heterozygosity in the outbred animals was 60·4%, 51·1% in the inbred animals and 0% in the gynogenetic clones. The average number of alleles among the eight animals was 4·7 per marker. Six markers (18·8%) gave an additional polymorphic amplification product besides the polymorphic amplification product in the expected size range. The possibility that these loci are tetraploid is discussed. The polymorphic loci described for common carp will be valuable as genetic markers for use in population, breeding, and evolutionary studies.     

Molecular cloning and characterization of common carp (Cyprinus carpio L.) TCRgamma and CD3gamma/delta chains

Partial cDNA sequences of TCRgamma and CD3gamma/delta were isolated from the thymus of common carp (Cyprinus carpio L.) by the method of suppression subtractive hybridization (SSH). Subsequently the full length cDNAs of carp TCRgamma and CD3gamma/delta were obtained by means of 3' RACE and 5' RACE, respectively. The full length of carp TCRgamma chain is 1368bp and encodes 326 amino acids including a signal peptide region of 19 amino acids and a transmembrane region of 23 amino acids at the C-terminal region from aa 291 to 313. The V region of carp TCRgamma contains 109 amino acids, the core motif FGXG in J segment was also found in carp TCRgamma. The C region of carp TCRgamma contains the characteristic CX6PX6WX45C motif. The CP region of carp TCR Cgamma contains 37 amino acids. The full length of carp CD3gamma/delta is 790bp and encodes 175 amino acids including a signal peptide region of 17 amino acids and a transmembrane region of 23 amino acids from aa 93 to 115. Similar to other known CD3gamma/deltas, four cysteine residues in the extracellular domain and an immunoreceptor tyrosine-based activation motif ITAM (YxxL/Ix6-8YxxL/I) in the intracellular domain are also included in carp CD3gamma/delta. Differing from other known CD3gamma/deltas, carp CD3gamma/delta lacks the CXXCXE motif in the extracellular domain. RT-PCR analysis demonstrated that the expression of TCRgamma gene was mainly in the thymus and gill of 6-month carp, but in 18-month carp, TCRgamma gene was detected in all the examined tissues. The expression of CD3gamma/delta gene was detected in all examined tissues of 6 and 18-month carp; among them, the highest expression level was in the thymus of 6-month carp. In situ hybridization showed that CD3gamma/delta-expressing cells were widely distributed in the head kidney, spleen and kidney of carp, whereas in the thymus, they were densely distributed in the lymphoid outer zone and scattered in the epithelioid inner zone.     

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.     

Molecular characteristics of a single and novel form of carp (Cyprinus carpio) monoamine oxidase

Two mammalian monoamine oxidases (MAO), MAO-A and MAO-B, are similar in primary structures but have unique substrate/inhibitor selectivities. Carp (Cyprinus carpio) contains a MAO enzyme (C-MAO) with properties different from MAO-A and MAO-B. To determine the molecular characteristics of C-MAO and its phylogenetic relationship with other fish and mammalian MAOs, the primary structure of C-MAO was estimated. The putative C-MAO cDNA encodes 526 amino acids with 59.001 Da, and the deduced amino acid sequence showed as much as 68.9% homology with some mammalian MAO-A proteins, 69.8% homology with some mammalian MAO-B proteins, and as much as 92.4% homology with some fish MAOs. Comparison of two regions in the polypeptide sequence of C-MAO determining possible substrate/inhibitor preferences of MAO-A and MAO-B showed both 79.5% homologies.     

Structure, organization and expression of common carp (Cyprinus carpio L.) SLP-76 gene

SLP-76 is an important member of the SLP-76 family of adapters, and it plays a key role in TCR signaling and T cell function. Partial cDNA sequence of SLP-76 of common carp (Cyprinus carpio L.) was isolated from thymus cDNA library by the method of suppression subtractive hybridization (SSH). Subsequently, the full length cDNA of carp SLP-76 was obtained by means of 3' RACE and 5' RACE, respectively. The full length cDNA of carp SLP-76 was 2007 bp, consisting of a 5'-terminal untranslated region (UTR) of 285 bp, a 3'-terminal UTR of 240 bp, and an open reading frame of 1482 bp. Sequence comparison showed that the deduced amino acid sequence of carp SLP-76 had an overall similarity of 34-73% to that of other species homologues, and it was composed of an NH2-terminal domain, a central proline-rich domain, and a C-terminal SH2 domain. Amino acid sequence analysis indicated the existence of a Gads binding site R-X-X-K, a 10-aa-long sequence which binds to the SH3 domain of LCK in vitro, and three conserved tyrosine-containing sequence in the NH2-terminal domain. Then we used PCR to obtain a genomic DNA which covers the entire coding region of carp SLP-76. In the 9.2k-long genomic sequence, twenty one exons and twenty introns were identified. RT-PCR results showed that carp SLP-76 was expressed predominantly in hematopoietic tissues, and was upregulated in thymus tissue of four-month carp compared to one-year old carp. RT-PCR and virtual northern hybridization results showed that carp SLP-76 was also upregulated in thymus tissue of GH transgenic carp at the age of four-months. These results suggest that the expression level of SLP-76 gene may be related to thymocyte development in teleosts.     

PCR-cloning and gene expression studies in common carp (Cyprinus carpio) insulin-like growth factor-II

Insulin-like growth factor-II (IGF-II) is a member of a growth factor family related to fetal growth in mammals but its physiological role has not been clearly identified in fish. In teleosts, the basic mechanism of the growth hormone (GH)-IGF axis is known to be operative but in a different manner. For instance, IGF-I exhibits GH dependence whereas for IGF-II, its GH dependence varies in different fish species. In this study, we used polymerase chain reaction (PCR) to obtain a common carp IGF-II (ccIGF-II) cDNA fragment and methods of rapid amplification of cDNA ends (RACEs) to obtain a full-length ccIGF-II sequence. The ccIGF-II encodes for a predicted amino acid sequence showing identities of 70.6%, 68.7%, 63.4% and 35% in comparison with salmon, barramundi, tilapia and human IGF-II, respectively. The nucleotide identity between the open reading frame (ORF) of the ccIGF-II and ccIGF-I cDNA sequence is only 36.2%. Distribution of ccIGF-II mRNA levels in common carp tissues was also studied; ccIGF-II expressed in hepatopancreas, heart, and many other tissues in adult carps are similar to the levels of ccIGF-I except in gills and testis. ccIGF-II levels were significantly higher than that of ccIGF-I in most juvenile tissues except in hepatopancreas, where ccIGF-I was higher (threefold) than that of ccIGF-II. The levels of ccIGF-I were also higher than ccIGF-II in carp larvae, from pre-hatched stage to day 30 post-hatching. Injection of porcine GH (pGH) increased the IGF-I and IGF-II mRNA levels in the hepatopancreas and brain of juvenile carps. However, hepatic IGF-I mRNA levels were induced more than IGF-II by pGH, whereas ccIGF-II levels gave a higher response than IGF-I in the brain in response to GH induction.     

Genetic control of glucosephosphate isomerase (GPI) in common carp (Cyprinus carpio L.)

In common carp, a freshwater fish species of tetraploid origin, GPI enzymes are present in two variants: GPI-A and GPI-B. GPI-A is coded by two loci segregating for two (GPI-A 1*) and six (GPI-A2*) alleles. Experimental crosses of the ornamental (Koi) variety of common carp revealed that GPI-B is coded by only one locus (GPI-B*). Another GPI-B* locus must have been silenced in the process of functional diploidization. It was also shown that the GPI-A2* locus segregated independently from the GPI-B* locus, demonstrating that the loci are located on different chromosomes.     

Genetic evolution and diversity of common carp Cyprinus carpio L.

Knowledge of genetic variation and population structure of existing strains of both farmed and wild common carp Cyprinus carpio L. is absolutely necessary for any efficient fish management and/or conservation program. To assess genetic diversity in common carp populations, a variety of molecular markers were analyzed. Of those, microsatellites and mitochondrial DNA were most frequently used in the analysis of genetic diversity and genome evolution of common carp. Using microsatellites showed that the genome evolution in common carp exhibited two waves of rearrangements: one whole-genome duplication (12–16 million years ago) and a more recent wave of segmental duplications occurring between 2.3 and 6.8 million years ago. The genome duplication event has resulted in tetraploidy since the common carp currently harbors a substantial portion of duplicated loci in its genome and twice the number of chromosomes (n = 100–104) of most other cyprinid fishes. The variation in domesticated carp populations is significantly less than that in wild populations, which probably arises from the loss of variation due to founder effects and genetic drift. Genetic differentiation between the European carp C.c. carpio and Asian carp C.c. haematopterus is clearly evident. In Asia, two carp subspecies, C.c. haematopterus and C.c. varidivlaceus, seem to be also genetically distinct.     

Polypeptide modulators of caspase recruitment domain (CARD)-CARD-mediated protein-protein interactions

The caspase recruitment domain (CARD) is present in a large number of proteins. Initially, the CARD was recognized as part of the caspase activation machinery. CARD-CARD interactions play a role in apoptosis and are responsible for the Apaf-1-mediated activation of procaspase-9 in the apoptosome. CARD-containing proteins mediate the inflammasome-dependent activation of proinflammatory caspase-1. More recently, new roles for CARD-containing proteins have been reported in signaling pathways associated with immune responses. The functional role of CARD-containing proteins and CARDs in coordinating apoptosis and inflammatory and immune responses is not completely understood. We have explored the putative cross-talk between apoptosis and inflammation by analyzing the modulatory activity on both the Apaf-1/procaspase-9 interaction and the inflammasome-mediated procaspase-1 activation of CARD-derived polypeptides. To this end, we analyzed the activity of individual recombinant CARDs, rationally designed CARD-derived peptides, and peptides derived from phage display.     

鲤鱼微卫星突变速率和模式(英文)

利用150个微卫星分子标记在F1代家系的基因型分析过程中,共有27600个等位基因从亲本向子代传递,其中在5个微卫星座位上检测到6个突变的等位基因。对突变的等位基因数目进行统计分析后得出:鲤鱼平均每个世代每个微卫星座位的突变速率为2.53×10-4。在发现突变的5个位点中,经测序发现,突变序列中插入1个以上的重复单元就导致了突变的发生。这些突变表明,鲤鱼的微卫星突变没有遵循严格的渐变突变模型(stepwise mutation model,SMM)。该文关于鲤鱼微卫星突变速率和模式的研究将会对统计鲤鱼有效群体的统计提供有效参数。