Chromosomal location of the 28S ribosomal RNA gene of channel catfish by in situ polymerase chain reaction

This study describes the use of the polymerase chain reaction for physical mapping of fish genes. A 287–base pair (bp) fragment of the 28S ribosomal RNA gene (28S rDNA) of channel catfish Ictalurus punctatus was isolated and sequenced with human-derived primers. The nucleotide (nt) sequence of this fragment was 20 bp shorter than that of the corresponding region of the human 28S rDNA. The gene was mapped to chromosomes of channel catfish by fluorescence in situ hybridization (FISH) and in situ polymerase chain reaction (ISPCR). A major locus and a minor locus of 28S rDNA were found on chromosomes of channel catfish. The major locus was associated with the active nucleolus organizer region (NOR) sites. The minor locus was highly resolved and not detectable by silver staining, suggesting that this locus was not involved in synthesis of ribosomal RNA and possessed fewer copies of 28S rDNA. Both loci contained GC-rich DNA elements that could be components of 28S rDNA repeated units. In this study, a potential method of comparative mapping of the channel catfish genome has been presented by using human-derived oligonucleotide sequences. These data demonstrate that ISPCR is highly specific and will be useful in physical mapping of fish genomes.     

Complete mitochondrial genome of the bullhead torrent catfish, Liobagrus obesus (Siluriformes, Amblycipididae): Genome description and phylogenetic considerations inferred from the Cyt b and 16S rRNA genes

Mitochondrial DNA (mtDNA) from the bullhead torrent catfish, Liobagrus obesus, was isolated by long-polymerase chain reaction (Long-PCR) with universal primers and was fully sequenced by primer working using flanking sequences. The complete mtDNA from L. obesus was 16,531 bp in length and contained 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and a control region, demonstrating a structure very similar to that of other bony fish. An analysis of the protein-coding genes revealed a statistically substantiated bias in (T+C): (A+G) content, supporting earlier findings regarding this peculiarity. As indicated by a chi-square test, the observed scores for pyrimidine and purine content were different from those expected assuming a 50:50 ratio: chi(2)=41.63, d.f.=5, p<0.000001 for three categories, including the 1st, 2nd, and 3rd codon positions. Further, there was a difference in nucleotide content between ND6 and the other 12 protein-coding genes in L. obesus. The values of p-distances, as summarized for different scales of evolutionary history at the Cyt b gene, revealed a clear pattern of increased nucleotide diversity at four levels: (1) intraspecies, (2) intragenus, (3) intrafamily, and (4) intraorder. Scores of average p-distances of the four categories in catfish were (1) 1.59+/-0.54%, (2) 5.28+/-1.72% (3) 16.37+/-1.26%, and (4) 19.81+/-0.14%, respectively. These data support the hypothesis that speciation in the order Siluriformes, in most cases, follows a geographic mode through the accumulation of a numerous small genetic changes over a long time period. A phylogenetic tree for the bullhead torrent catfish and several other fish species belonging to the order Siluriformes was developed on the basis of respective Cyt b sequences (1138 bp); the analysis revealed a monophyletic origin for the five examined families. A species-specific clustering of sequences from single species was obtained, supporting additionally basic phylogenetic information for the catfish and the barcoding suitability of Cyt b sequence data. Lastly, one of the well-supported properties of our phylogenetic tree (99% repetition level in our analysis) was the monophyletic placement of all catfish (order Siluriformes) among other ray-finned fish of the class Actinopterigii. Also discussed herein are the aspects of phylogeny based on the 16S rRNA gene.     

Development of Polymorphic EST Markers Suitable for Genetic Linkage Mapping of Catfish

Expressed sequence tag (EST) markers are important for gene mapping and for marker-assisted selection (MAS). To develop EST markers for use in catfish gene mapping, 100 randomly picked complementary DNAs from the channel catfish (Ictalurus punctatus) pituitary library were sequenced. The EST sequences were used to design primers to amplify channel catfish and blue catfish (I. furcatus) genomic DNAs. Polymerase chain reaction products of the ESTs were analyzed to determine length polymorphism between the channel catfish and blue catfish. Eleven polymorphic EST markers were identified. Five of the 11 EST markers were from known genes and the other six were from unidentified ESTs. Seven ESTs were found to be associated with microsatellite sequences. Analysis of channel catfish gene sequences indicated highly biased codon usage, with 16 codons being preferably used. These codons were more preferably used in highly expressed ribosomal protein genes and in highly expressed pituitary hormone genes. G/C-rich codons are less used in channel catfish than those in other vertebrates suggesting AT-richness of the channel catfish genome. Received June 29, 1998; accepted March 29, 1999.     

Immunoglobulin heavy chain constant and heavy chain variable region genes in phylogenetically diverse species of bony fish

Summary Genomic DNA from 18 phylogenetically diverse species of bony fish was hybridized with probes specific for the channel catfish immunoglobulin heavy chain constant (CH) gene, as well as with immunoglobulin heavy chain variable (VH) probes specific for five channel catfish VH gene families. The results showed that CH probes strongly hybridized only to genomic fragments from other catfish species. In contrast, restricted DNA from most other species hybridized with at least two channel catfish VH probes. In those species whose DNA hybridized with multiple VH probes, the restriction pattern of hybridizing fragments was probe-dependent. These studies suggest that (1) the CH gene defined in channel catfish appears to share similarity only with CH genes in other catfish species, (2) families of VH genes appear to have diverged in early phylogenetic lineages of teleosts, and (3) VH genes similar to those defined in catfish appear to be widely represented in phylogenetically diverse species of teleosts.     

Genomic organization and evolution of olfactory receptors and trace amine-associated receptors in channel catfish,Ictalurus punctatus

BackgroundChannel catfish (Ictalurus punctatus) live in turbid waters with limited visibility to chase prey within a certain distance. This can be compensated through detecting specific water-soluble substances by the olfactory receptors (ORs) and trace amine associated receptors (TAARs) expressed on the olfactory epithelium.MethodsWe identified the OR and TAAR repertoires in channel catfish, and characterized the genomic organizations of these two gene families by data mining available genomic resources.ResultsA total of 47 putative OR genes and 36 putative TAAR genes were identified in the channel catfish genome, including 27 functional OR genes and 28 functional TAAR genes. Phylogenetic and orthogroup analyses were conducted to illustrate the evolutionary dynamics of the vertebrate ORs and TAARs. Collinear analysis revealed the presence of two conserved orthologous blocks that contain OR genes between the catfish genome and zebrafish genome. The complete loss of a conserved motif in fish OR family H may contribute to the divergence of family H from other families. The dN/dS analysis indicated that the highest degree of selection pressure was imposed on TAAR subfamily 14 among all fish ORs and TAARs.ConclusionsThe present study provides understanding of the evolutionary dynamics of the two gene families (OR and TAAR) associated with olfaction in channel catfish.General significanceThis is the first systematic study of ORs and TAARs in catfish, which could provide valuable genomic resources for further investigation of olfactory mechanisms in teleost fish.     

Urokinase is required for the formation of mactinin,an alpha-actinin fragment that promotes monocyte/macrophage maturation

Myostatin is a recently discovered gene that inhibits muscle growth. In the present study, we characterized the myostatin locus and its expression in channel catfish (Ictalurus punctatus). The genomic DNA and cDNA encoding the channel catfish myostatin were cloned and sequenced. The myostatin gene has three exons encoding a protein of 389 amino acids. Comparison of the genomic sequences with those of the cDNA revealed that the myostatin cDNA was 1673 base pair (bp) long with a 5'-untranslated region (UTR) and 3'-UTR of 180 and 323 bp, respectively. The deduced amino acid sequences of the catfish myostatin is highly conserved with those of other organisms. The myostatin locus is highly polymorphic in channel catfish because of the presence of several microsatellites and single nucleotide polymorphic sites. The myostatin gene was expressed in various tissues and developmental stages at differential levels, suggesting complex regulation of this gene and perhaps roles for myostatin in addition to those originally suggested.     

Nucleotide sequence of channel catfish heavy chain cDNA and genomic blot analyses. Implications for the phylogeny of Ig heavy chains

Our prior analyses defined the cDNA sequence on part of the CH2 domain, the complete CH3 and CH4 domains, and the 3'-untranslated region of a catfish H chain. To complete the catfish H chain mRNA sequence, a primer-extended H chain cDNA library was constructed. Analysis of this library has resulted in the definition of full-length clones encoding a 61-bp 5' untranslated region, a 51-bp leader sequence, the V region and the complete CH1 and CH2 domains. The high similarity defined with other vertebrate V regions readily allowed the catfish sequence to be divided into FR and CDR regions. Sequence comparisons with mammalian VH and JH genes strongly suggest that the catfish V region is the product of multiple genes. Using a catfish VH cDNA probe, at least 25 different genomic VH members were defined. Because this probe does not hybridize with other full-length H chain cDNA clones, additional VH families will likely be defined in catfish. Phylogenetic sequence comparisons of the catfish C region domains indicated that the CH1 and CH4 were the most highly conserved. In addition several important features were defined in genomic Southern blot analyses of catfish DNA. Gene titration experiments established that the catfish CH gene is represented by a single genomic copy. This finding provides clear evidence that the genomic organization of H chain genes in catfish must be different from that defined in sharks and suggests that the phylogeny of single copy CH genes may have been established at the level of the bony fishes. It is also likely that there is an additional CH gene in catfish. This gene is also represented by a single genomic copy, and based upon its relative signal intensity when compared with the known CH gene it appears to share higher similarity with the known CH1 domain than it does with the CH2 domain.     

Gene structure of the carp fish ribosomal protein L41: seasonally regulated expression

The seasonal acclimatization of the carp fish demands physiological compensatory responses. The process involves profound nucleolar adjustments and remarkable changes in rRNA synthesis, which affect ribosomal biogenesis. We have documented that protein kinase CK2, whose activity is related to ribosomal protein L41 and the regulation of rRNA synthesis, was expressed in notably higher amounts in summer-acclimatized carp compared to the cold-season adapted fish. Thus, we approached the study of the functional genomics of carp L41 protein. We report the first cloning of a fish L41 gene encoding the highly conserved 25 amino acids, including approximately 1700 bp regulatory upstream region and the 3(') polyadenylation signal, plus the isolation and characterization of two different L41 cDNAs. We found a clear differential expression of L41, which follows the same pattern as protein kinase CK2beta that transcribes at higher levels in the summer-acclimatized carp than it does in the winter-adapted fish.     

黄颡鱼Wnt家族4个基因的克隆、组织表达及对铜的响应

为探究Wnt家族成员在黄颡鱼卵巢发育中的作用, 研究首先采用RT-PCR和RACE技术获取了黄颡鱼Wnt5a、Wnt5b、Wnt7a和Wnt9b基因的全长cDNA序列, 即1984、2905、2158和1622 bp, 其中ORF长度分别为1124、1124、1049和1073 bp, 编码375、375、350和358个氨基酸。氨基酸序列比对和系统树分析显示, 这些基因十分保守, 黄颡鱼WNT与墨西哥丽脂鲤和斑马鱼比较接近。组织表达分析表明, 这些基因的mRNA在脑、脾脏、肾脏、鳃、心脏、肌肉、脂肪、肝脏及卵巢等组织中都有表达, 但表达水平不尽相同。Wnt家族基因对铜的响应研究表明: 在暴露28d时, Wnt7a mRNA水平随着铜浓度先上升后下降, 但是Wnt5a、Wnt5b和Wnt9b基因表达各个处理组无显著性差异; 在56d, Wnt5b在60 μg Cu/L组最低, 其他2个组差异不显著, Wnt9b mRNA水平随着铜浓度先上升后下降, 但是Wnt5a和Wnt7a基因表达各个处理组无显著性差异, 表明Wnt家族这些基因的功能发生了分化, 部分成员介导了铜影响黄颡鱼卵巢发育的调控。研究首次揭示了黄颡鱼Wnt家族部分成员的基因结构和功能, 为深入探讨他们在卵巢发育中的作用奠定了基础。     

Ribosomal protein L4 of Saccharomyces cerevisiae: the gene and its protein.

The sequence of a gene for ribosomal protein L4 of Saccharomyces cerevisiae has been determined. Unlike most ribosomal protein genes of S. cerevisiae this gene has no intron. The single open reading frame predicts that L4 is highly homologous to mammalian ribosomal protein L7a. There appear to be two genes for L4, both of which are active.     

Cloning and characterization of myogenic regulatory genes in three Ictalurid species

We report sequence, tissue expression and map-position data for myogenin, MYOD1, myostatin and follistatin in three Ictalurid catfish species: channel catfish (Ictalurus punctatus), blue catfish (I. furcatus) and white catfish (Ameiurus catus). These genes are involved in muscle growth and development in mammals and may play similar roles in catfish. Amino acid sequences were highly conserved among the three Ictalurid species (>95% identity), moderately conserved among catfish and zebrafish (approximately 80% identity), and less conserved among catfish and humans (approximately 40-60% identity) for all four genes. Gene structure (number of exons and introns and exon-intron boundaries) was conserved between catfish and other species for all genes. Myogenin and MYOD1 expression was limited to skeletal muscle in juvenile channel catfish, similar to expression patterns for these genes in other fish and mammalian species. Myostatin was expressed in a variety of tissues in juvenile channel catfish, a pattern common in other fish species but contrasting with data from mammals where myostatin is primarily expressed in skeletal muscle. Follistatin was expressed in juvenile catfish heart, testes and spleen. All four genes contained polymorphic microsatellite repeats in non-coding regions and linkage analysis based on inheritance of these microsatellite loci was used to place the genes on the channel catfish linkage map. Information provided in this study will be useful in further studies to determine the role these genes play in muscle growth and development in catfish.     

Piscine glycoprotein hormone (gonadotropin and thyrotropin) receptors: a review of recent developments

Similar to the higher vertebrates, the pituitary in bony fishes express three glycoprotein hormones: thyroid-stimulating hormone (TSH), follicle-stimulating hormone (FSH) and luteinizing hormone (LH). In addition to the appropriate secretion of these hormones, the timely and quantitative expression of their specific receptors (TSHR, FSHR and LHR) in the target tissues is an essential requirement for their physiological action. In fishes that constitute a very diverse group of vertebrates, there are only a few published reports of primary structure of these receptors although other examples have been communicated briefly. This review will summarize these reports as well as to describe the insights gained from what is known about the mammalian receptors. The structural organization of the fish receptors (as deduced from the encoding cDNAs) is highly homologous to the higher vertebrate receptors in that there is a 7-pass transmembrane region and an N-terminal extracellular domain, which contributes to ligand specificity. In mammals, the FSHR and the TSHR genes are composed of 10 exons whereas the LHR gene is composed of 11 exons. The position of the 'extra intron' is conserved in the catfish LHR gene. In the mammals, the transmembrane domain of each of the three glycoprotein hormone receptors is encoded by a single exon, however, in the salmon genes and homologous invertebrate genes, this portion of the receptor is encoded by multiple exons. In general, the tissue-specific expression of these receptors is similar to that seen in mammals, however, the gonadal expression of TSHR in the striped bass and sunrise sculpin and the renal expression of LHR in the channel catfish are unique.     

Molecular responses of ceruloplasmin to Edwardsiella ictaluri infection and iron overload in channel catfish (Ictalurus punctatus)

Ceruloplasmin is a serum ferroxidase that carries more than 90% of the copper in plasma and has documented roles in iron homeostasis as well as antioxidative functions. In our previous studies, it has been shown that the ceruloplasmin gene is strongly up-regulated in catfish during challenge with Edwardsiella ictaluri. However, little is known about the function of this gene in teleost fish. The objective of this study, therefore, was to characterize the ceruloplasmin gene from channel catfish, determine its genomic organization, profile its patterns of tissue expression, and establish its potential for physiological antioxidant responses in catfish after bacterial infection with E. ictaluri and iron treatment. The genomic organization suggested that the catfish ceruloplasmin gene had 20 exons and 19 introns, encoding 1074 amino acids. Exon sizes of the catfish ceruloplasmin gene were close to or identical with mammalian and zebrafish homologs. Further phylogenetic analyses suggested that the gene was highly conserved through evolution. The catfish ceruloplasmin gene was mapped to both the catfish physical map and linkage map. The catfish ceruloplasmin gene was mainly expressed in liver with limited expression in other tissues, and it was significantly up-regulated in the liver after bacterial infection alone or after co-injection with bacteria and iron-dextran, while expression was not significantly induced with iron-dextran treatment alone.     

Simultaneous horizontal gene transfer of a gene coding for ribosomal protein l27 and operational genes in Arthrobacter sp

Phylogenetic analysis of bacterial L27 ribosomal proteins showed that, against taxonomy, the L27 protein from the Actinobacteria Arthrobacter sp. clusters with protein sequences from the Bacillus group. The L27 gene clusters in the Arthrobacter sp. genome with six genes responsible for creatinine and sarcosine degradation. Phylogenetic analyses of orthologue proteins encoded by three of these genes also showed a phylogenetic relationship with Bacillus species. Comparisons between the synonymous codon usage of the Arthrobacter sp. genes and those from complete genomes showed that Arthrobacter genes encoding the L27 ribosomal protein and the proteins responsible for the degradation of creatinine and sarcosine have a codon usage that is more similar to that of Bacillus species than that of Arthrobacter. We suggest that the Arthrobacter sp. genes encoding the L27 ribosomal protein and the proteins responsible for the degradation of creatinine and sarcosine were acquired simultaneously through horizontal gene transfer from an unknown Bacillus species.     

核糖体蛋白RPL15 cDNA序列在真骨鱼类系统进化研究中的价值 &

应用RT-PCR, 从真骨总目（Teleostei）5目15种鱼类中首次克隆了核糖体大亚基蛋白L15 (RPL15, ribosomal protein L15) 的完整cDNA序列。以海鲢形亚组（Elopomorpha）的鳗鲡作为外类群, 对这些真骨鱼类的核糖体蛋白L15 cDNA序列进行了系统发育分析, 结果表明: (1)RPL15基因在真骨鱼类等许多真核生物进化中高度保守; (2)系统树中各物种之间的关系与形态分类一致。RPL15编码区适合于真骨鱼类目以上分类阶元的分子系统学研究。