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A.P12 Molecular analysis of the immunoglobulin Vh repertoire in the rainbow trout
The Scientific and Social Program of the V1~h ISDCI Congress
A.Pll DIVERSITY TROUT
OF THE IMMUNOGLOBULIN
VII R E P E R T O I R E
$51
IN THE RAINBOW
Thibaud Roman, Jacques Charlemagne University P. et M. Curie, CNRS, URA 1135, Groupe d'Immunologie Compar6e, 9 Quai Saint Bernard, 75252 Paris Cedex 05, France A VHcDNA library was made by anchored PCR from the head kidney cytoplasmic RNA of a 8.5 monthold non immunized rainbow trout. Six new V. segments were characterized from this library, thus bringing to nine the number of V H families actually defined in that species. A phylogenic analysis indicates that the nine V. families can be classified in three major groups. The first one includes the VHI, VH3, V,4 and V,7 families and is homologous to the human and mouse Group III VH families (as defined by Tutter and Riblet, PNAS, 86:7460,1989). The second group include the V.5, V,8 and V.9 families and is much related to the Group I and Group II human and mouse families. The third group is represented by the V.2 and VH6 families which are not closely related to any other vertebrate V.. An extensive analysis of the VDJ heavy chain junctions was undertaken by PCR from the head kidney RNA of 15 month-old non immunized trouts. Over the 74 analyzed VDJ segments, 72 different sequences were detected, thus suggesting an important structural diversity of natural antibodies. Six different JH segments were determined and two of them (JHB and JHD) are overrepresented, thus introducing a biais in the JH usage of the sample analyzed. At least 8 VM segments were used in the sample and possible D-D fusions were observed. Although few informations are available on the germinal structure of the rainbow trout genomic D. and Ja elements, our analysis strongly suggests that an important level of N-diversity occurrs at the VH-DH and DH-JH junctions.
A.P12 MOLECULAR ANALYSIS THE RAINBOW TROUT
OF THE
IMMUNOGLOBULIN
VII R E P E R T O I R E
IN
Thibaud Roman, Jacques Charlemagne University P. et M. Curie, CNRS, URA 1135, Groupe d'Immunologie Compar6e, 9 Quai SaintBernard, 75252 Paris Cedex 05, France Total cytoplasmic RNA from the head kidney cells of a 8.5 month-old non immunized rainbow trout was used for the confection of a V. library by anchored PCR. From this library, 47 clones were completely or partially sequenced and none was more than 75% similar to the previously described trout VH segments. Six new V H families were characterized, thus bringing to nine the number of V, families actually characterized in the rainbow trout. All these new V~ segments exhibit the canonical features of known vertebrate V., including the conserved Cys22, Cys92 and Trp36. A phylogenic analysis indicates that the nine V H families can be classified in three major groups. The first one includes the V.1, V.3, V,4 and V~7 families and is homologous to the human and mouse Group III V. families (as defined by Tutter and Riblet, PNAS, 86: 7460, 1989). The second group include the V,5, V.8 and V.9 families and is much related to the Group 1 and Group II human and mouse families. The third group is represented by the V.2 and V,6 families which are not closely related to any other vertebrate V.. Six different JH segments were characterized, temporary designated JHA to J,F, the J.F segments corresponding to the more 3' segments located 825 bp upstream to the C~tA exon (Lee et al., Molec. lmmunol., 30: 641, 1993). Over the 72 sequences analyzed, the JHA and JHC segments are used only once and two J~ [JHB (23/72) and JHD (26/72)] are overrepresented, thus introducing a biais in the JH usage in the sample. The phylogenic conservation of the trout JH structure is evident, including the canonical Trpl03, Glyl04, (X105), Glyl06, Thrl07 stretch wich is present in most vertebrate JH sequences.
A.Pll DIVERSITY TROUT
OF THE IMMUNOGLOBULIN
VII R E P E R T O I R E
$51
IN THE RAINBOW
Thibaud Roman, Jacques Charlemagne University P. et M. Curie, CNRS, URA 1135, Groupe d'Immunologie Compar6e, 9 Quai Saint Bernard, 75252 Paris Cedex 05, France A VHcDNA library was made by anchored PCR from the head kidney cytoplasmic RNA of a 8.5 monthold non immunized rainbow trout. Six new V. segments were characterized from this library, thus bringing to nine the number of V H families actually defined in that species. A phylogenic analysis indicates that the nine V. families can be classified in three major groups. The first one includes the VHI, VH3, V,4 and V,7 families and is homologous to the human and mouse Group III VH families (as defined by Tutter and Riblet, PNAS, 86:7460,1989). The second group include the V.5, V,8 and V.9 families and is much related to the Group I and Group II human and mouse families. The third group is represented by the V.2 and VH6 families which are not closely related to any other vertebrate V.. An extensive analysis of the VDJ heavy chain junctions was undertaken by PCR from the head kidney RNA of 15 month-old non immunized trouts. Over the 74 analyzed VDJ segments, 72 different sequences were detected, thus suggesting an important structural diversity of natural antibodies. Six different JH segments were determined and two of them (JHB and JHD) are overrepresented, thus introducing a biais in the JH usage of the sample analyzed. At least 8 VM segments were used in the sample and possible D-D fusions were observed. Although few informations are available on the germinal structure of the rainbow trout genomic D. and Ja elements, our analysis strongly suggests that an important level of N-diversity occurrs at the VH-DH and DH-JH junctions.
A.P12 MOLECULAR ANALYSIS THE RAINBOW TROUT
OF THE
IMMUNOGLOBULIN
VII R E P E R T O I R E
IN
Thibaud Roman, Jacques Charlemagne University P. et M. Curie, CNRS, URA 1135, Groupe d'Immunologie Compar6e, 9 Quai SaintBernard, 75252 Paris Cedex 05, France Total cytoplasmic RNA from the head kidney cells of a 8.5 month-old non immunized rainbow trout was used for the confection of a V. library by anchored PCR. From this library, 47 clones were completely or partially sequenced and none was more than 75% similar to the previously described trout VH segments. Six new V H families were characterized, thus bringing to nine the number of V, families actually characterized in the rainbow trout. All these new V~ segments exhibit the canonical features of known vertebrate V., including the conserved Cys22, Cys92 and Trp36. A phylogenic analysis indicates that the nine V H families can be classified in three major groups. The first one includes the V.1, V.3, V,4 and V~7 families and is homologous to the human and mouse Group III V. families (as defined by Tutter and Riblet, PNAS, 86: 7460, 1989). The second group include the V,5, V.8 and V.9 families and is much related to the Group 1 and Group II human and mouse families. The third group is represented by the V.2 and V,6 families which are not closely related to any other vertebrate V.. Six different JH segments were characterized, temporary designated JHA to J,F, the J.F segments corresponding to the more 3' segments located 825 bp upstream to the C~tA exon (Lee et al., Molec. lmmunol., 30: 641, 1993). Over the 72 sequences analyzed, the JHA and JHC segments are used only once and two J~ [JHB (23/72) and JHD (26/72)] are overrepresented, thus introducing a biais in the JH usage in the sample. The phylogenic conservation of the trout JH structure is evident, including the canonical Trpl03, Glyl04, (X105), Glyl06, Thrl07 stretch wich is present in most vertebrate JH sequences.