Two cathelicidin genes are present in both rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar)

Further to the previous finding of the rainbow trout rtCATH_1 gene, this paper describes three more cathelicidin genes found in salmonids: two in Atlantic salmon, named asCATH_1 and asCATH_2, and one in rainbow trout, named rtCATH_2. All the three new salmonid cathelicidin genes share the common characteristics of mammalian cathelicidin genes, such as consisting of four exons and possessing a highly conserved preproregion and four invariant cysteines clustered in the C-terminal region of the cathelin-like domain. The asCATH_1 gene is homologous to the rainbow trout rtCATH_1 gene, in that it possesses three repeat motifs of TGGGGGTGGC in exon IV and two cysteine residues in the predicted mature peptide, while the asCATH_2 gene and rtCATH_2 gene are homologues of each other, with 96% nucleotide identity. Salmonid cathelicidins possess the same elastase-sensitive residue, threonine, as hagfish cathelicidins and the rabbit CAP18 molecule. The cleavage site of the four salmonid cathelicidins is within a conserved amino acid motif of QKIRTRR, which is at the beginning of the sequence encoded by exon W. Two 36-residue peptides corresponding to the core part of rtCATH_1 and rtCATH_2 were chemically synthesized and shown to exhibit potent antimicrobial activity. rtCATH_2 was expressed constitutively in gill, head kidney, intestine, skin and spleen, while the expression of rtCATH_1 was inducible in gill, head kidney, and spleen after bacterial challenge. Four cathelicidin genes have now been characterized in salmonids and two were identified in hagfish, confirming that cathelicidin genes evolved early and are likely present in all vertebrates.

Isolation and characterization of microsatellites in Chinese sturgeon, Acipenser sinensis

From (GATA)(n) and (AAAG)(n) enriched genomic libraries for the Chinese sturgeon (Acipenser sinensis), 50 primer pairs were developed using the fast isolation by AFLP of sequences containing repeats (FIASCO) protocol. Forty-six primer pairs exhibited highly polymorphic with two to 11 alleles per locus, while the rest four displayed monomorphic. These markers yielded 246 alleles in a survey of eight specimens of wild A. sinensis. Average observed heterozygosity ranged from 0.13 to 1.00. These loci should provide sufficient levels of genetic diversity to allow parentage analysis for artificial stocking management and delineation of fine-scale population structure.

Gene cloning and functional analysis of glycosaminoglycan-degrading enzyme chondroitin AC lyase from Flavobacterium columnare G(4)

The chondroitin AC lyase gene, cslA, was cloned for the first time from the fish bacterial pathogen F. columnare G(4). From the first transcription initiation site, the cslA extends 2620 nucleotides to the end of the 3' region. The open reading frame of cslA transcript has 2286 nucleotides encoding 762 amino acids with a 16 residues long signal peptide at the N-terminus. The gene, cslA was then successfully expressed in Escherichia coli and recombinant chondroitin AC lyase, rChonAC was purified, with its lytic activity analyzed. Zymography analysis copolymerized with chondroitin sulphate revealed the lytic activity of rChonAC and also the crude native ChonAC isolated from periplamic space of cultured F. columnare G(4). The low level of lytic activity observed in crude native ChonAC may be due possibly to the low level of expression of this gene in the cultured condition. The expression and the role of this virulence factor is of interest for further research on the pathogenesis of F. columnare.

Identification of a novel cathelicidin gene in the rainbow trout, Oncorhynchus mykiss

We report the cloning of a novel antimicrobial peptide gene, termed rtCATH_1, found in the rainbow trout, Oncorhynchus mykiss. The predicted 216-residue rtCATH_1 prepropeptide consists of three domains: a 22-residue signal peptide, a 128-residue cathelin-like region containing two identifiable cathelicidin family signatures, and a predicted 66-residue C-terminal cationic antimicrobial peptide. This predicted mature peptide was unique in possessing features of different known (mammalian) cathelicidin subgroups, such as the cysteine-bridged family and the specific amino-acid-rich family. The rtCATH_1 gene comprises four exons, as seen in all known mammalian cathelicidin genes, and several transcription factor binding sites known to be of relevance to host defenses were identified in the 5' flanking region. By Northern blot analysis, the expression of rtCATH_1 was detected in gill, head kidney, and spleen of bacterially challenged fish. Primary cultures of head kidney leukocytes from rainbow trout stimulated with lipopolysaccharide or poly(I (.) C) also expressed riCATH_1. A 36-residue peptide corresponding to the core part of the fish cathelicidin was chemically synthesized and shown to exhibit potent antimicrobial activity and a low hemolytic effect. Thus, rtCATH_1 represents a novel antimicrobial peptide gene belonging to the cathelicidin family and may play an important role in the innate immunity of rainbow trout.

Identification of immune genes in grass carp Ctenopharyngodon idella in response to infection of the parasitic copepod Sinergasilus major

The parasitic copepod Sinergasilus major is an important pathogen of grass carp Ctenopharyngodon idella. To understand the immune response of grass carp to the copepod infection, suppression subtractive hybridization method was employed to characterize genes up-regulation during the copepod infection in liver and gills of the fish. One hundred and twenty-two dot blot positive clones from infected subtracted library were sequenced. Searching available databases by using these nucleotide sequences revealed that 23 genes are immune-related, including known acute-phase reactants, and four novel genes encoding proteins such as source of immunodominant MHC-associated peptides (SIMP), TNF receptor-associated factor 2 binding protein (T2BP), poliovirus receptor-related protein 1 precursor, glycoprotein A repetitions predominant (GARP). The differential expression of seven immune genes, i.e. GARP, alpha-2-macroglobulin, MHC class I, C3, SIMP, T2BP, transferrin, as a result of infection was further confirmed by RT-PCR, with the up-regulation of alpha-2-macroglobulin, MHC class I, C3, SIMP and T2BP in the liver of infected fish, and down-regulation of SIMP in the gills of infected fish. The present study provides foundation for understanding grass carp immune response and candidate genes for further analysis.

Amplified fragment length polymorphism analysis to identify the genetic structure of the Gymnocypris przewalskii (Kessler, 1876) population from the Qinghai Basin, China

Amplified fragment length polymorphism (AFLP) was used to analyse the genetic structure of 45 individuals of Gymnocypris przewalskii (Kessler, 1876), an endangered and state-protected rare fish species, from three areas [the Heima (HM), Buha (BH) and Shaliu rivers (SL), all draining into Qinghai Lake]. A total of 563 polymorphic loci were detected. The HM, BH and SL populations have 435, 433 and 391 loci, respectively (Zhu and Wu, 1975), which account for 77.26%, 76.91% and 69.45% of the total number of polymorphic loci of each population, respectively. The Nei indices of genetic diversities (H) of the three populations were calculated to be 0.2869 (HM), 0.2884 (BH) and 0.2663 (SL), respectively. Their Shannon informative indices are 0.4244, 0.4251 and 0.3915, respectively. Research results show that the mean genetic distance between HM and BH is the smallest (0.0511), between BH and SL is the second shortest (0.0608), and between HM and SL is the largest (0.0713), with the mean genetic distance among the three populations being over 0.05. Data mentioned above indicate that the three populations have a certain genetic differentiation. The total genetic diversity (H-t = 0.3045) and the mean value of genetic diversity within the population (H-s = 0.2786) indicate that the variations have mainly come from within the population.

Molecular cloning of TRAF2 binding protein gene and its promoter region from the grass carp Ctenopharyngodon idellus

A tumor necrosis factor receptor-associated factor 2 binding protein (T2BP) gene was isolated from the grass carp (Ctenopharyngodon idellus) by utilizing suppression subtractive hybridization (SSH) and rapid amplification of cDNA ends (RACE). The grass carp T2BP (GT2BP) gene contains an open reading frame of 579 nucleotide(s) (nt), encoding 193 amino acids, with 23 nt 5'-untranslated region and a long 3'-untranslated region of 434 nt including poly (A), 1 AUUUA motif and 4 AUUUUA motifs. No signal peptide has been detected in the predicted GT2BP, but a characteristic forkhead associated domain is present. The GT2BP mRNA shares 83% identity with the zebrafish DNA sequence, and they both have no introns in the genomic DNA. The putative transcription factor binding sites of GT2BP include two C/EBP alpha binding sites, and one c-Jun binding, one AP-1 binding, and one nuclear factor kappa B (NF kappa B) binding sites. Southern blot analysis revealed that the GT2BP was a single-copy gene. Individual difference was observed in GT2BP expression in examined organs of healthy grass carp. However, the expression of GT2BP in all examined organs in a fish with the highest copepod infection level and the significantly higher expression level in spleen and liver in infected fish may indicate its up-regulation with the parasite infection. (c) 2005 Elsevier B.V. All rights reserved.

Linichthys: A new genus of Chinese cyprinid fishes (Teleostei : Cypriniformes)

yLinichthys, new genus, is proposed for Barbodes laticeps Lin and Zhang, 1986. It can be separated from all other genera of Asian Barbinae in having a shallow depression extending along the median portion of the upper lip. Linichthys is superficially very similar to the type species of a monotypic Chinese cyprinid cavefish genus, Typhlobarbus nudiventris, in having an elongate and somewhat compressed body form, a truncate distal margin of the dorsal fin, and a soft and smooth last unbranched dorsal-fin ray, all of them being atypical for most of Asian barbins. The two genera differ in size of the eye and the number of rows of pharyngeal tooth. Although the same body form and/or structure of the dorsal fin are also shared with some species currently placed in other genera of Asian barbins, the recognition of Linichthys is warranted because of their marked differences in other characters. A redescription is also provided for the type species of the monotypic genus, Linichthys laticeps. It is known only from the upper and middle Zhu Jiang basin and the upper Chang Jiang basin in the city of Guiyang, Guizhou Province, South China.

Complex mutation at a microsatellite locus in sturgeons: Acipenser sinensis, A-schrenckii, A-gueldenstaedtii and A-baerii

Cross-species amplifications of microsatellite locus Spl-106, which was originally screened from the genome of shovelnose sturgeon (Scaphirhynchus platorynchus) with a perfect TAGA repeat motif, were carried out in four other species of the genera Acipenser. A total of 34 polymerase chain reaction (PCR) products representing 16 different alleles of this locus was sequenced. Sequence analysis results showed that besides the number changes of repeat units, many mutational events, such as single-base substitutions and various insertion/deletion (indels) occurred not only at species level but also at individual level, even among the different alleles within the same individual. The repeat motifs varied from perfect (TAGA)n array to perfect compound (TAAA)m (GAAA)n and perfect or imperfect compound (TAAA)m (TAGA)n (TAAA)x arrays in different species and different individuals. The evolution dynamics of this locus in sturgeons was inferred in that it may evolve from a single perfect to different perfect or imperfect compounds.

Microsatellites assessment of Chinese sturgeon (Acipenser sinensis Gray) genetic variability

Four microsatellites were used to examine the genetic variability of the spawning stocks of Chinese sturgeon, Acipenser sinensis, from the Yangtze River sampled over a 3-year period (1999-2001). Within 60 individuals, a total of 28 alleles were detected over four polymorphic microsatellite loci. The number of alleles per locus ranged from 4 to 15, with an average allele number of 7. The number of genotypes per locus ranged from 6 to 41. The genetic diversity of four microsatellite loci varied from 0.34 to 0.67, with an average value of 0.54. For the four microsatellite loci, the deviation from the Hardy-Weinberg equilibrium was mainly due to null alleles. The mean number of alleles per locus and the mean heterozygosity were lower than the average values known for anadromous fishes. Fish were clustered according to their microsatellite characteristics using an unsupervised 'Artificial Neural Networks' method entitled 'Self-organizing Map'. The results revealed no significant genetic differentiation considering genetic distance among samples collected during different years. Lack of heterogeneity among different annual groups of spawning stocks was explained by the complex age structure (from 8 to 27 years for males and 12 to 35 years for females) of Chinese sturgeon, leading to formulate an hypothesis about the maintenance of genetic diversity and stability in long-lived animals.

Characterization of two genes encoding leucine-rich repeat-containing proteins in grass carp Ctenopharyngodon idellus

The cDNAs and genes of two different types of leucine- rich repeat-containing proteins from grass carp ( Ctenopharyngodon idellus) were cloned. Homology search revealed that the two genes, designated as GC-GARP and GC-LRG, have 37% and 32% deduced aminoacid sequence similarities with human glycoprotein A repetitions predominant precursor ( GARP) and leucine-rich alpha2-glycoprotein (LRG), respectively. The cDNAs of GC-GARP and GC-LRG encoded 664 and 339 amino acid residues, respectively. GC-GARP and GC-LRG contain many distinct structural and/or functional motifs of the leucine- rich repeat (LRR) subfamily, such as multiple conserved 11-residue segments with the consensus sequence LxxLxLxxN/CxL ( x can be any amino acid). The genes GC-GARP and GC-LRG consist of two exons, with 4,782 bp and 2,119 bp in total length, respectively. The first exon of each gene contains a small 5'-untranslated region and partial open reading frame. The putative promoter region of GC-GARP was found to contain transcription factor binding sites for GATA-1, IRF4, Oct-1, IRF-7, IRF-1, AP1, GATA-box and NFAT, and the promoter region of GC-LRG for MYC-MAX, MEIS1, ISRE, IK3, HOXA9 and C/EBP alpha. Phylogenetic analysis showed that GC-GARP and mammalian GARPs were clustered into one branch, while GC-LRG and mammalian LRGs were in another branch. The GC-GARP gene was only detected in head kidney, and GC-LRG in the liver, spleen and heart in the copepod ( Sinergasilus major)- infected grass carp, indicating the induction of gene expression by the parasite infection. The results obtained in the present study provide insight into the structure of fish LRR genes, and further study should be carried out to understand the importance of LRR proteins in host - pathogen interactions.

Characterization of development-related genes for the cestode Bothriocephalus acheilognathi

Differential gene expression of mature and immature Bothriocephalus acheilognathi cestodes was analyzed using the suppression subtractive hybridization technique. Five mature-associated cDNAs were isolated and characterized. Virtual Northern blot and RT-PCR analyses confirmed that four of the five genes were up-regulated in mature parasites. The sequence analysis revealed that one gene encoded the structural protein chorion precursor, and that three encoded functional proteins homologous to yolk ferritin, sodium/hydrogen exchanger and muscin-like protein. Another gene appeared to be specific to B. acheilognathi, encoding a putative metal-bound protein. Although results obtained in the present study are preliminary, the information about the five genes may provide clues for further investigation on the decline in parasite numbers during the maturation of B. acheilognathi.

Discovery and characterization of two types of liver-expressed antimicrobial peptide 2 (LEAP-2) genes in rainbow trout

The sequences and gene organisation of two LEAP-2 molecules (LEAP-2A and LEAP-2B) from rainbow trout, Oncorhynchus mykiss are presented. Both genes consist of a 3 exon/2 intron structure, with exon sizes comparable to known mammalian genes. LEAP-2A notably differs from LEAP-2B in having larger introns and a larger 3'UTR. The predicted proteins contain a signal peptide and prodomain, followed by a mature peptide of 41 aa containing four conserved cysteines. The RXXR cleavage site to release the mature peptide was also conserved. Both genes were found to be constitutively expressed in the liver, with expression in the intestine, and to a lesser extent the skin, evident after bacterial challenge. (C) 2004 Elsevier B.V. All rights reserved.

Conservation strategies for endemic fish species threatened by the Three Gorges Dam

The largest damming project to date, the Three Gorges Dam has been built along the Yangtze River (China), the most species-rich river in the Palearctic region. Among 162 species of fish inhabiting the main channel of the upper Yangtze, 44 are endemic and are therefore under serious threat of global extinction from the dam. Accordingly, it is urgently necessary to develop strategies to minimize the impacts of the drastic environmental changes associated with the dam. We sought to identify potential reserves for the endemic species among the 17 tributaries in the upper Yangtze, based on presence/absence data for the 44 endemic species. Potential reserves for the endemic species were identified by characterizing the distribution patterns of endemic species with an adaptive learning algorithm called a "self-organizing map" (SOM). Using this method, we also predicted occurrence probabilities of species in potential reserves based on the distribution patterns of communities. Considering both SOM model results and actual knowledge of the biology of the considered species, our results suggested that 24 species may survive in the tributaries, 14 have an uncertain future, and 6 have a high probability of becoming extinct after dam filling.

HLA-DRB1 allele polymorphisms in genetic susceptibility to esophageal carcinoma

AIM: To probe into the genetic susceptibility of HLA-DRB1 alleles to esophageal carcinoma in Han Chinese in Hubei Province. METHODS: HLA-DRB1 allele polymorphisms were typed by polymerase chain reaction with sequence-specific primers (PCR-SSP) in 42 unrelated patients with esophageal cancer and 136 unrelated normal control subjects and the associated HLA-DRB1 allele was measured by nucleotide sequence analysis with PCR.SAS software was used in statistics. RESULTS: Allele frequency (AF) of HLA-DRB1*0901 was significantly higher in esophageal carcinoma patients than that in the normal controls (0.2500 vs0.1397, P=0.028, the odds ratio 2.053, etiologic fraction 0.1282). After analyzed the allele nucleotide sequence of HLA-DRB1*0901 which approachs to the corresponded exon 2 sequence of the allele in genebank. There was no association between patients and controls in the rested HLA-DRB1 alleles. CONCLUSION: HLA-DRB1*0901 allele is more common in the patients with esophageal carcinoma than in the healthy controls, which is positively associated with the patients of Hubei Han Chinese. Individuals carrying HLA-DRB1*0901 may be susceptible to esophageal carcinoma.