The cDNAs coding for the α- and β-subunits of Xenopus laevis casein kinase II

AbstractUsing a λgt10 cDNA library obtained from Xenopus laevis oocytes and probes derived from the known sequences of the human and Drosophila genes, a cDNA coding for the α-subunit of the X. laevis casein kinase II was isolated. The coding sequence of this clone determines a polypeptide of 350 amino acids. The X. laevis sequence is 98% identical to the human and rat proteins in the first 323 amino acids. Using the polymerase chain reaction to generate a 370-nucleotide-long probe, it was possible to clone and sequence a cDNA of 900 nucleotides that coded for the X. laevis β-subunit of casein kinase II. The derived protein sequence is 215 amino acids long and again shows an extraordinary degree of conservation with other species

Genome-Wide Association Analysis for Resistance to Infectious Pancreatic Necrosis Virus Identifies Candidate Genes Involved in Viral Replication and Immune Response in Rainbow Trout (Oncorhynchus mykiss)

Infectious pancreatic necrosis (IPN) is a viral disease with considerable negative impact on the rainbow trout (Oncorhynchus mykiss) aquaculture industry. The aim of the present work was to detect genomic regions that explain resistance to infectious pancreatic necrosis virus (IPNV) in rainbow trout. A total of 2,278 fish from 58 full-sib families were challenged with IPNV and 768 individuals were genotyped (488 resistant and 280 susceptible), using a 57K SNP panel Axiom, Affymetrix. A genome-wide association study (GWAS) was performed using the phenotypes time to death (TD) and binary survival (BS), along with the genotypes of the challenged fish using a Bayesian model (Bayes C). Heritabilities for resistance to IPNV estimated using genomic information, were 0.53 and 0.82 for TD and BS, respectively. The Bayesian GWAS detected a SNP located on chromosome 5 explaining 19% of the genetic variance for TD. The proximity of Sentrin-specific protease 5 (SENP5) to this SNP makes it a candidate gene for resistance against IPNV. In case of BS, a SNP located on chromosome 23 was detected explaining 9% of the genetic variance. However, the moderate-low proportion of variance explained by the detected marker leads to the conclusion that the incorporation of all genomic information, through genomic selection, would be the most appropriate approach to accelerate genetic progress for the improvement of resistance against IPNV in rainbow trout

Whole Genome Linkage Disequilibrium and Effective Population Size in a Coho Salmon (Oncorhynchus kisutch) Breeding Population Using a High-Density SNP Array

The estimation of linkage disequilibrium between molecular markers within a population is critical when establishing the minimum number of markers required for association studies, genomic selection, and inferring historical events influencing different populations. This work aimed to evaluate the extent and decay of linkage disequilibrium in a coho salmon breeding population using a high-density SNP array. Linkage disequilibrium was estimated between a total of 93,502 SNPs found in 64 individuals (33 dams and 31 sires) from the breeding population. The markers encompass all 30 coho salmon chromosomes and comprise 1,684.62 Mb of the genome. The average density of markers per chromosome ranged from 48.31 to 66 per 1 Mb. The minor allele frequency averaged 0.26 (with a range from 0.22 to 0.27). The overall average linkage disequilibrium among SNPs pairs measured as r2 was 0.10. The Average r2 value decreased with increasing physical distance, with values ranging from 0.21 to 0.07 at a distance lower than 1 kb and up to 10 Mb, respectively. An r2 threshold of 0.2 was reached at distance of approximately 40 Kb. Chromosomes Okis05, Okis15 and Okis28 showed high levels of linkage disequilibrium (>0.20 at distances lower than 1 Mb). Average r2 values were lower than 0.15 for all chromosomes at distances greater than 4 Mb. An effective population size of 43 was estimated for the population 10 generations ago, and 325, for 139 generations ago. Based on the effective number of chromosome segments, we suggest that at least 74,000 SNPs would be necessary for an association mapping study and genomic predictions. Therefore, the SNP panel used allowed us to capture high-resolution information in the farmed coho salmon population. Furthermore, based on the contemporary Ne, a new mate allocation strategy is suggested to increase the effective population size

CK2α/CK1α chimeras are sensitive to regulation by the CK2β subunit

The effect of CK2β on the activity of CK2α and other protein kinases that can bind this regulatory subunit is not fully understood. In an attempt to improve our understanding of this effect, chimeras of CK2α and CK1α have been constructed. These chimeras contain different portions of the CK2α amino terminal region that are involved in the interaction with CK2β to form CK2 tetramers. In the case of chimeras 1 and 2, the portions of CK2α replace the corresponding segments of CK1α. In the case of chimera 3, the fragment of CK2α is added to the whole CK1α molecule with the exception of the initial methionine. Chimera 3 has 8% of the activity of CK1αWT, while chimeras 1 and 2 are 3 orders of magnitude less active than CK1αWT. All three chimeras bind tightly to CK2β, but only chimeras 1 and 2 are significantly stimulated in their capacity to phosphorylate casein and canonical peptide substrates by addition of the regulatory subunit. No stimulation was observed with phosvitin or non-canonica

Autophosphorylation of carboxy-terminal residues inhibits the activity of protein kinase CK1α

CK1 constitutes a protein kinase subfamily that is involved in many important physiological processes. However, there is limited knowledge about mechanisms that regulate their activity. Isoforms CK1δ and CKlε were previously shown to autophosphorylate carboxy-terminal sites, a process which effectively inhibits their catalytic activity. Mass spectrometry of CKla and splice variant CKlαL has identified the autophosphorylation of the last four carboxyl-end serines and threonines and also for CKlαS, the same four residues plus threonine-327 and serine-332 of the S insert. Autophosphorylation occurs while the recombinant proteins are expressed in Escherichia coli. Mutation of four carboxy-terminal phosphorylation sites of CKlα to alanine demonstrates that these residues are the principal but not unique sites of autophosphorylation. Treatment of autophosphorylated CKla and CKlaS with λ phosphatase causes an activation of 80-100% and 300%, respectively. Similar treatment fails to stimulate

Molecular detection and species identification of Alexandrium (Dinophyceae) causing harmful algal blooms along the Chilean coastline

© The Authors 2012. Background and aims On the basis of morphological evidence, the species involved in South American Pacific coast harmful algal blooms (HABs) has been traditionally recognized as Alexandrium catenella (Dinophyceae). However, these observations have not been confirmed using evidence based on genomic sequence variability. Our principal objective was to accurately determine the species of Alexandrium involved in local HABs in order to implement a real-time polymerase chain reaction (PCR) assay for its rapid and easy detection on filter-feeding shellfish, such as mussels. Methodology For species-specific determination, the intergenic spacer 1 (ITS1), 5.8S subunit, ITS2 and the hypervariable genomic regions D1-D5 of the large ribosomal subunit of local strains were sequenced and compared with two data sets of other Alexandrium sequences. Speciesspecific primers were used to amplify signature sequences within the genomic DNA of the studied species by conventional and real

Assessing footprints of selection in commercial Atlantic salmon populations using microsatellite data

Artículo de publicación ISI.Relatively large rates of response to traits of economic importance have been observed in different selection experiments in salmon. Several QTL have been mapped in the salmon genome, explaining unprecedented levels of phenotypic variation. Owing to the relatively large selection intensity, individual loci may be indirectly selected, leaving molecular footprints of selection, together with increased inbreeding, as its likely relatives will share the selected loci. We used population differentiation and levels of linkage disequilibrium in chromosomes known to be harbouring QTL for body weight, infectious pancreatic necrosis resistance and infectious salmon anaemia resistance to assess the recent selection history at the genomic level in Atlantic salmon. The results clearly suggest that the marker SSA0343BSFU on chromosome 3 (body weight QTL) showed strong evidence of directional selection. It is intriguing that this marker is physically mapped to a region near the coding sequence of DVL2 , making it an ideal candidate gene to explain the rapid evolutionary response of this chromosome to selection for growth in Salmo salar. Weak evidence of diversifying selection was observed in the QTL associated with infectious pancreatic necrosis and infectious salmon anaemia resistance. Overall, this study showed that artificial selection has produced important changes in the Atlantic salmon genome, validating QTL in commercial salmon populations used for production purposes according to the recent selection history.CONICYT (FONDECYT 1090632)

Activity of Recombinant α and β Subunits of Casein Kinase II from Xenopus laevis

Casein kinase II (CKII) is a ubiquitous protein kinase, found predominantly in cell nuclei, which has two subunits in a tetrameric α2β2 or αα′2 conformation. The catalytic center is present in the α subunit which is active by itself while β is a regulatory subunit that can greatly enhance the activity of α. The cDNA genes of Xenopus laevis coding for the α and β subunits of CKII have been expressed in Escherichia coli and extensively purified. The recombinant subunits reconstitute a fully active holoenzyme when incubated in stoichiometric amounts. Mutations that change serines in positions 2 and 3 of the β subunit for glycines completely eliminate the autophosphorylation site present in this subunit but do not significantly affect the capacity of β to activate α. A fusion protein composed of glutathione transferase linked to the X. laevis CKII β subunit can also activate α. This fusion protein binds to glutathione-agarose beads and can mediate the binding of the α subunit to this matr

Data from: Genomic predictions and genome-wide association study of resistance against Piscirickettsia salmonis in coho salmon (Oncorhynchus kisutch) using ddRAD sequencing

Piscirickettsia salmonis is one of the main infectious diseases affecting coho salmon (Oncorhynchus kisutch) farming, and current treatments have been ineffective for the control of this disease. Genetic improvement for P. salmonis resistance has been proposed as a feasible alternative for the control of this infectious disease in farmed fish. Genotyping by sequencing (GBS) strategies allow genotyping of hundreds of individuals with thousands of single nucleotide polymorphisms (SNPs), which can be used to perform genome wide association studies (GWAS) and predict genetic values using genome-wide information. We used double-digest restriction-site associated DNA (ddRAD) sequencing to dissect the genetic architecture of resistance against P. salmonis in a farmed coho salmon population and to identify molecular markers associated with the trait. We also evaluated genomic selection (GS) models in order to determine the potential to accelerate the genetic improvement of this trait by means of using genome-wide molecular information. A total of 764 individuals from 33 full-sib families (17 highly resistant and 16 highly susceptible) were experimentally challenged against P. salmonis and their genotypes were assayed using ddRAD sequencing. A total of 9,389 SNPs markers were identified in the population. These markers were used to test genomic selection models and compare different GWAS methodologies for resistance measured as day of death (DD) and binary survival (BIN). Genomic selection models showed higher accuracies than the traditional pedigree-based best linear unbiased prediction (PBLUP) method, for both DD and BIN. The models showed an improvement of up to 95% and 155% respectively over PBLUP. One SNP related with B-cell development was identified as a potential functional candidate associated with resistance to P. salmonis defined as DD