Characterization of two POU transcription factor family members from the urochordate Oikopleura dioica.

Three POU domain containing transcription factors have been cloned from the urochordate Oikopleura dioica. Phylogenetic analysis showed that two of these (OctA1 and OctA2) are closely related members of the class II POU domain family, and one (OctB) is a member of the class III POU domain family. All three transcription factors contained a highly conserved bipartite DNA-binding POU domain with POU specific and POU homeodomains, separated by a linker region. All three proteins were shown to bind specifically to the canonical octamer motif, ATGCAAAT. The ability of these factors to drive transcription from an octamer-containing reporter construct was assessed in vertebrate B lymphocyte cell lines. Both OctA1 and OctA2 drove transcription in murine and catfish B cell lines, however, OctB did not increase the level of transcription above background levels. It is concluded that Oct transcription factors capable of functioning in a similar fashion to vertebrate Oct1/2 were present at the phylogenetic level of the urochordates.

New resources for marine genomics: bacterial artificial chromosome libraries for the Eastern and Pacific oysters (Crassostrea virginica and C. gigas).

Large-insert genomic bacterial artificial chromosome (BAC) libraries of two culturally and economically important oyster species, Crassostrea virginica and C. gigas, have been developed as part of an international effort to develop tools and reagents that will advance our ability to conduct genetic and genomic research. A total of 73,728 C. gigas clones with an average insert size of 152 kb were picked and arrayed representing an 11.8-fold genome coverage. A total of 55,296 clones with an average insert size of 150 kb were picked and arrayed for C. virginica, also representing an 11.8-fold genome coverage. The C. gigas and C. virginica libraries were screened with probes derived from selected oyster genes using high-density BAC colony filter arrays. The probes identified 4 to 25 clones per gene for C. virginica and 5 to 50 clones per gene for C. gigas. We conducted a preliminary analysis of genetic polymorphism represented in the C. gigas library. The results suggest that the degree of divergence among similar sequences is highly variable and concentrated in intronic regions. Evidence supporting allelic polymorphism is reported for two genes and allelic and/or locus specific polymorphism for several others. Classical inheritance studies are needed to confirm the nature of these polymorphisms. The oyster BAC libraries are publicly available to the research community on a cost-recovery basis at (www.genome.clemson.edu).

The antibody site in Atlantic salmon; phage display and modeling of scFv with anti-hapten binding ability.

Cloning and sequencing the cDNA of around 50 VH (VDJ) and 15 VL genes in Atlantic salmon demonstrated nine VH families (above 80% identity within each family) and one dominating but relatively diverse VL family in this species. The highest variability of the VH was seen in the CDR3, but CDR2 also expressed a modest variability. The 'whole' antibody repertoire was expressed as single chain Fv (scFv) in a phage display library by combining 12 VH and two VL specific primers (FR1/microl and FR1/CL, respectively). The PCR products (VH and VL) were ligated (with a G-rich spacer) into the lambda Surf-Zap (Stratagene) vector and expressed as a surface fusion protein on the M13 phage. Anti-TNP and anti-FITC specific scFv clones were isolated by panning using hapten-coated magnetic beads and the coding DNA sequenced. The specificities of the anti-TNP and anti-FITC clones were similar to mouse monoclonal antibodies. 3D-models of the active sites (CDRs) of the anti-TNP and anti-FITC clones suggest hapten-interacting structures of the salmon antibody site similar to mammalian antibodies.