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

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.

Structural interpretation of site-directed mutagenesis and specificity of the catalytic subunit of protein kinase CK2 using comparative modelling.

The catalytic subunit of protein kinase casein kinase 2 (CK2alpha), which has specificity for both ATP and GTP, shows significant amino acid sequence similarity to the cyclin-dependent kinase 2 (CDK2). We constructed site-directed mutants of CK2alpha and used a three-dimensional model to investigate the basis for the dual specificity. Introduction of Phe and Gly at positions 50 and 51, in order to restore the pattern of the glycine-rich motif, did not seriously affect the specificity for ATP or GTP. We show that the dual specificity probably originates from the loop situated around the position His115 to Asp120 (HVNNTD). The insertion of a residue in this loop in CK2 alpha subunits, compared with CDK2 and other kinases, might orient the backbone to interact with the base A and G; this insertion is conserved in all known CK2alpha. The mutant deltaN118, the design of which was based on the modelling, showed reduced affinity for GTP as predicted from the model. Other mutants were intended to probe the integrity of the catalytic loop, alter the polarity of a buried residue and explore the importance of the carboxy terminus. Introduction of Arg to replace Asn189, which is mapped on the activation loop, results in a mutant with decreased k(cat), possibly as a result of disruption of the interaction between this residue and basic residues in the vicinity. Truncation at position 331 eliminates the last 60 residues of the alpha subunit and this mutant has a reduced catalytic efficiency compared with the wild-type. Catalytic efficiency is restored in the truncation mutant by the replacement of a potentially buried Glu at position 252 by Lys, probably owing to a higher stability resulting from the formation of a salt bridge between Lys252 and Asp208.

Activity of the E75E76 mutant of the alpha subunit of casein kinase II from Xenopus laevis.

The cDNA gene coding for the alpha subunit of Xenopus laevis casein kinase II was mutated using the overlap extension PCR method. The mutation substituted glutamic acids for Lys75 and Lys76, changing the charge distribution of a very basic sequence found in the alpha subunit. Expression of the mutated cDNA in a pT7-7 vector in E. coli yielded an active mutant recombinant protein that was extensively purified. This mutant was not significantly affected in its app. Km for casein or a model peptide substrate, nor in its interaction with the activating beta subunit. Inhibition by quercetin and by 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole was also the same for mutant and wild type subunits. However, the CKII alpha E75E76 mutant was at least one order of magnitude less sensitive to inhibition by polyanionic inhibitors such as heparin, poly U, copolyglutamic acid:tyrosine (4:1) and 2,3 diphosphoglycerate.

Activity of recombinant alpha and beta 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 alpha 2 beta 2 or alpha alpha' beta 2 conformation. The catalytic center is present in the alpha subunit which is active by itself while beta is a regulatory subunit that can greatly enhance the activity of alpha. The cDNA genes of Xenopus laevis coding for the alpha and beta 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 beta subunit for glycines completely eliminate the autophosphorylation site present in this subunit but do not significantly affect the capacity of beta to activate alpha. A fusion protein composed of glutathione transferase linked to the X. laevis CKII beta subunit can also activate alpha. This fusion protein binds to glutathione-agarose beads and can mediate the binding of the alpha subunit to this matrix. Conversely, the alpha subunit was found to bind to glass fiber filters in an active form that can still be activated by beta to an extent similar to that seen in solution. Using peptides containing tyrosine and glutamic acid as inhibitors of the activity of the isolated alpha subunit and of the holoenzyme, the effect of beta on the specificity of inhibition was studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of metal ions on the activity of casein kinase II from Xenopus laevis.

Casein kinase II purified from the nuclei of Xenopus laevis oocytes as well as the recombinant alpha and beta subunits of the X. laevis CKII, produced in E. coli from the cloned cDNA genes, were tested with different divalent metal ions. The enzyme from both sources was active with either Mg2+, Mn2+, or Co2+. Optimal concentrations were 7-10 mM for Mg2+, 0.5-0.7 mM for Mn2+ and 1-2 mM for Co2+. In the presence of Mn2+ or Co2+ the enzyme used GTP more efficiently than ATP as a phosphate donor while the reverse was true in the presence of Mg2+. The apparent Km values for both nucleotide triphosphates were greatly decreased in the presence of Mn2+ as compared with Mg2+. Addition of Zn2+ (above 150 microM) to an assay containing the optimal Mg2+ ion concentration caused strong inhibition of both holoenzyme and alpha subunit. Inhibition of the holoenzyme by 400 microM Ni2+ could be reversed by high concentrations of Mg2+ but no reversal of this inhibition was observed with the alpha subunit.

The cDNAs coding for the alpha- and beta-subunits of Xenopus laevis casein kinase II.

Using a lambda gt10 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 alpha-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 beta-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.

The gamete membrane fusion test to assay the fertilizing ability of human spermatozoa.

The value of the gamete fusion test for the assessment of human spermatozoa is assessed. The factors influencing the test are discussed, including the conditions of heterologous fertilization using zona-free oocytes such as the nature of sperm preparation, sperm concentration and capacitation time, and the importance of albumin in the medium. The oocytes can be examined before or after fixation, and the spermatozoa around the eggs assessed for acrosome changes, while the number of sperm tails on the oocyte can be related to the number of chromatin decondensations. The test provides information on the fertilizing ability of spermatozoa of given individuals, and may prove of value in testing contraceptives.

Human sperm-cervical mucus interaction and the ability of spermatozoa to fuse with zona-free hamster oocytes.

Samples of semen and cervical mucus were provided by 18 couples. Cervical mucus was obtained for each day possible and stored at 4 degrees C until all the samples were collected. Flat capillary tubes were loaded with the mucous samples and spermatozoa from the husband's semen sample were allowed to migrate through the cervical mucus (3 cm column) into culture medium. The spermatozoa recovered after migration through cervical mucus were assayed in vitro with zona-free hamster oocytes. Control experiments were carried out using spermatozoa from the same semen sample but prepared by the swimming-up technique. Altogether, 557 eggs in the control group and 1236 eggs in the experimental group were analysed, and the results demonstrated that the % of sperm penetration, the mean number of sperm decondensations per penetrated egg and the mean number of spermatozoa adhering per egg all had higher values (P less than 0.05) for the control samples than for the experimental samples. We suggest that cervical mucus modifies human spermatozoa, as measured by their interaction with zona-free hamster oocytes.

Relationship between sperm concentration and the rate of fertilization in vitro of golden hamster eggs

El éxito de la fecundación depende de una interacción gamética adecuada. La evidencia disponible sugiere que el espermatozoide atravesará la zona pelúcida sólo cuando se asocia con ella en foram oblicua a través de sus bordes dorsal/ventral y anterior. Esta asociación es mediada por receptores espermáticos presentes en el borde externo de la zona pelúcida. La eficiencia de la penetración espermática podría estar asociada con la posibilidad que el espermatozoide pueda establecer ese tipo de asociación. Esta hipótese de trabajo se ensayó con un estudio realizado in vitro en el cual ovocitos de hamster con zona pelúcida se inseminaron con espermatozoides capacitados a concentraciones de 1.000, 24.111 y 48.000 espermatozoides/µl. Los resultados mostraron que al aumentar la concentración espermática, la tasa de fecundación bajó de un 97.7%, con la concentración espermática más baja, a un 63% con la concentración más alta. De la misma manera se observó que el promedio de 2,77 cuando se usó la concentració más alta. En ambos casos las diferencias fueron estadísticamente significativas (P < 0.001). Las observaciones con el microscopio electrónico de barrido mostraron que al usar concentraciones bajas de espermatozoides, éstos se asociaban con la zona pelúcida por sus bordes dorsa/ventral y anterior. Al usar concentraciones altas de espermatozoides, éstos se asociaban a través de su borde anterior. En el primer caso la asociación resultaba en una penetración exitosa, en cambio en el segundo caso, muchos espermatozoides eran incapaces de cruzar la zona

Relationship between sperm concentration and the rate of fertilization in vitro of golden hamster eggs

El éxito de la fecundación depende de una interacción gamética adecuada. La evidencia disponible sugiere que el espermatozoide atravesará la zona pelúcida sólo cuando se asocia con ella en foram oblicua a través de sus bordes dorsal/ventral y anterior. Esta asociación es mediada por receptores espermáticos presentes en el borde externo de la zona pelúcida. La eficiencia de la penetración espermática podría estar asociada con la posibilidad que el espermatozoide pueda establecer ese tipo de asociación. Esta hipótese de trabajo se ensayó con un estudio realizado in vitro en el cual ovocitos de hamster con zona pelúcida se inseminaron con espermatozoides capacitados a concentraciones de 1.000, 24.111 y 48.000 espermatozoides/Al. Los resultados mostraron que al aumentar la concentración espermática, la tasa de fecundación bajó de un 97.7%, con la concentración espermática más baja, a un 63% con la concentración más alta. De la misma manera se observó que el promedio de 2,77 cuando se usó la concentració más alta. En ambos casos las diferencias fueron estadísticamente significativas (P < 0.001). Las observaciones con el microscopio electrónico de barrido mostraron que al usar concentraciones bajas de espermatozoides, éstos se asociaban con la zona pelúcida por sus bordes dorsa/ventral y anterior. Al usar concentraciones altas de espermatozoides, éstos se asociaban a través de su borde anterior. En el primer caso la asociación resultaba en una penetración exitosa, en cambio en el segundo caso, muchos espermatozoides eran incapaces de cruzar la zona (AU)

Role of sulfatide on phosphoenzyme formation and ouabain binding of the (Na+ + K+)ATPase.

A microsomal fraction rich in (Na+ + K+)ATPase activity has been isolated from the outer medulla of pig kidney. The ability of this preparation to form phosphoenzyme on incubation with [gamma-32P]ATP and to bind [3H]ouabain was studied when its sulfatide was hydrolyzed by arylsulfatase treatment. The K+-dependent hydrolysis of the Na+-dependent phosphorylated intermediate as well as the ouabain binding were inactivated in direct relation to the breakdown of sulfatide. Both characteristics of the (Na+ + K+)ATPase preparation, lost by arylsulfatase treatment, were partially restored by the sole addition of sulfatide. These experiments indicate that sulfatide may play a role in sodium ion transport either in the conformational transition of the K+-insensitive phosphointermediate, E1P, to the K+-sensitive intermediate, E2P, or in the configuration of the high-affinity binding site for K+ of the E2P form. In addition, this glycolipid may have a specific role in the proteolipidic subunit that binds ouabain.