Integrated genetic and morphological data support eco-evolutionary divergence of Angolan and South African populations of Diplodus hottentotus.

The genus Diplodus presents multiple cases of taxonomic conjecture. Among these the D. cervinus complex was previously described as comprising three subspecies that are now regarded as separate species: Diplodus cervinus, Diplodus hottentotus and Diplodus omanensis. Diplodus hottentotus exhibits a clear break in its distribution around the Benguela Current system, prompting speculation that Angolan and South African populations flanking this area may be isolated and warrant formal taxonomic distinction. This study reports the first integrated genetic [mitochondrial (mt)DNA and nuclear microsatellite] and morphological (morphometric, meristic and colouration) study to assess patterns of divergence between populations in the two regions. High levels of cytonuclear divergence between the populations support a prolonged period of genetic isolation, with the sharing of only one mtDNA haplotype (12 haplotypes were fully sorted between regions) attributed to retention of ancestral polymorphism. Fish from the two regions were significantly differentiated at a number of morphometric (69·5%) and meristic (46%) characters. In addition, Angolan and South African fish exhibited reciprocally diagnostic colouration patterns that were more similar to Mediterranean and Indian Ocean congeners, respectively. Based on the congruent genetic and phenotypic diversity we suggest that the use of hottentotus, whether for full species or subspecies status, should be restricted to South African D. cervinus to reflect their status as a distinct species-like unit, while the relationship between Angolan and Atlantic-Mediterranean D. cervinus will require further demo-genetic analysis. This study highlights the utility of integrated genetic and morphological approaches to assess taxonomic diversity within the biogeographically dynamic Benguela Current region.

Secondary contact and asymmetrical gene flow in a cosmopolitan marine fish across the Benguela upwelling zone.

The combination of oceanographic barriers and habitat heterogeneity are known to reduce connectivity and leave specific genetic signatures in the demographic history of marine species. However, barriers to gene flow in the marine environment are almost never impermeable which inevitably allows secondary contact to occur. In this study, eight sampling sites (five along the South African coastline, one each in Angola, Senegal and Portugal) were chosen to examine the population genetic structure and phylogeographic history of the cosmopolitan bluefish (Pomatomus saltatrix), distributed across a large South-east Atlantic upwelling zone. Molecular analyses were applied to mtDNA cytochrome b, intron AM2B1 and 15 microsatellite loci. We detected uncharacteristically high genetic differentiation (FST 0.15-0.20; P<0.001) between the fish sampled from South Africa and the other sites, strongly influenced by five outlier microsatellite loci located in conserved intergenic regions. In addition, differentiation among the remaining East Atlantic sites was detected, although mtDNA indicated past isolation with subsequent secondary contact between these East Atlantic populations. We further identified secondary contact, with unidirectional gene flow from South Africa to Angola. The directional contact is likely explained by a combination of the northward flowing offshore current and endogenous incompatibilities restricting integration of certain regions of the genome and limiting gene flow to the south. The results confirm that the dynamic system associated with the Benguela current upwelling zone influences species distributions and population processes in the South-east Atlantic.

Activation of pp60c-src transforming potential by mutations altering the structure of an amino terminal domain containing residues 90-95.

The overexpression of the c-src gene product, pp60c-src, in avian and rodent embryo cells is not sufficient to induce cellular transformation. In this study we report that structural alterations within an amino terminal domain of pp60c-src, the exon 3 domain (residues 84-115) activate the oncogenic potential of the c-src gene product. Site-directed mutagenesis of the c-src gene was used to generate c-src variants encoding pp60c-src proteins with the following amino acid alterations: tyr 90 to phe (pm90F); tyr 92 to phe (pm92F); arg 95 to either trp, lys, glu or gln (pm95W, 95K, 95E and 95Q, respectively), and deletion of residues 92-95 (dl92). C-src variants encoding proteins with the alteration of arg 95 to trp, glu, or lys, or containing the deletion of residues 92-95, induced alterations in cell morphology and promoted growth in soft agar as well as changes in glucose transport and in vivo tyrosine phosphorylation of cellular proteins (including calpactin I heavy chain, p36). Analysis of in vivo phosphorylation of the transforming variant src proteins revealed little detectable alteration in the phosphorylation of tyr 527, a putative site of tyrosine kinase regulation. Our results suggest that structural alterations within a domain distal to the catalytic (kinase) domain activate pp60c-src kinase activity and, concomitantly, oncogenic potential. Furthermore, we suggest that the exon 3 domain of pp60c-src may contribute to the regulation and/or substrate specificity of the c-src protein.

Epitope mapping of monoclonal antibodies to gag protein p19 of avian sarcoma and leukaemia viruses.

We have characterized a set of 15 monoclonal antibodies to p19gag, one of the internal proteins of avian sarcoma and leukaemia viruses. All the antibodies work in immune precipitations as well as in immunoblotting, though with different efficiencies. We have developed a simple epitope mapping technique, which uses partial chemical cleavages at methionine or tryptophan residues followed by immunoblotting from SDS-polyacrylamide gels, to localize the epitopes of nine of these antibodies. The epitopes fall into at least four classes. The mapping procedure should also be useful for other antigens of known primary structure.

A simple method for immunoaffinity purification of nondenatured avian sarcoma and leukemia virus gag-containing proteins.

We have developed a one-step purification procedure for proteins containing the N-terminal portion of the gag protein of avian sarcoma and leukemia viruses. In this procedure, a resin with a covalently attached monoclonal antibody to the gag protein p19 is used to bind gag-containing proteins from crude extracts. After washing of the resin, the bound proteins are eluted with 2 M MgCl2. For the transforming protein kinase encoded by Fujinami sarcoma virus p130gag-fps, this procedure gave an enrichment of several thousand-fold, a yield of over 10%, a final purity of over 20%, and no significant loss of protein kinase activity. Similar purifications were obtained with three other gag-containing proteins. The immunoaffinity purification described may be of general utility as a first step in purification of the several other avian retroviral transforming proteins that are synthesized from fusions of an oncogene with the viral gag gene.

Activation of the oncogenic potential of the avian cellular src protein by specific structural alteration of the carboxy terminus.

The role of tyrosine phosphorylation in the regulation of tyrosine protein kinase activity was investigated using site-directed mutagenesis to alter the structure and environment of the three tyrosine residues present in the C terminus of avian pp60c-src. Mutations that change Tyr 527 to Phe or Ser activate in vivo tyrosine protein kinase activity and induce cellular transformation of chicken cells in culture. In contrast, alterations of tyrosine residues present at positions 511 or 519 in c-src do not induce transformation or in vivo tyrosine protein kinase activity. Amber mutations, which alter the structure of the pp60c-src C terminus by inducing premature termination of the c-src protein at either residue 518 or 523 also induce morphological transformation and increase in vivo tyrosine phosphorylation, whereas removal of the last four residues of c-src by chain termination at residue 530 does not alter the kinase activity or the biological activity of the resultant c-src protein. We conclude from these studies that C-terminal alterations which either remove or replace Tyr 527 serve to activate the c-src protein resulting in cellular transformation and increased in vivo tyrosine protein kinase activity.