RESUMEN
Sequences of Aotus nancymaae immunoglobulin kappa light-chain rearrangements were analyzed after reverse transcription polymerase chain reaction. Among 22 in-frame rearrangements analyzed, 12 IGKV genes belonging to the families 1, 2, or 3 were identified. Aotus counterparts for all five human IGKJ genes were found. The identity of the deduced human and Aotus amino acid sequences was between 83% and 92% for junctional regions and 74% for the constant region. Sequence comparisons between rearrangements indicated that somatic mutations, the addition of nongermline-encoded nucleotides, and exonuclease trimming contribute to the generation of diversity of Aotus immunoglobulin kappa chains. The high identity of Aotus and human IGK genes is comparable to that of T-cell receptor genes and further supports the proposal to use the Aotus Plasmodium falciparum infection model for the evaluation of malaria vaccine candidates.
Asunto(s)
Aotidae/genética , Regiones Constantes de Inmunoglobulina/genética , Región de Unión de la Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/genética , Secuencia de Aminoácidos , Animales , Aotidae/inmunología , Secuencia de Bases , ADN Complementario , Humanos , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoRESUMEN
The Mexican axolotl V(H) segments associated with the Igh C mu and C nu isotypes were isolated from anchored PCR libraries prepared from spleen cell cDNA. The eight new V(H) segments found bring the number of V(H) families in the axolotl to 11. Each V(H) had the canonical structural features of vertebrate V(H) segments, including residues important for the correct folding of the Ig domain. The distribution of ser AGC/T (AGY) and TCN codons in axolotl V(H) genes was biased toward AGY in complementarity-determining region-1 (CDR1) and TCN in framework region-1 (FR1); there were no ser residues in the FR2 region. Thus, the axolotl CDR1 region is enriched in DNA sequences forming potential hypermutation hot spots and is flanked by DNA sequences more resistant to point mutation. There was no significant bias toward AGY in CDR2. Southern blotting using family-specific V(H) probes showed restriction fragments from 1 (V(H)9) to 11-19 (V(H)2), and the total number of V(H) genes was 44 to 70, depending on the restriction endonuclease used. The V(H) segments were not randomly used by the H mu and H nu chains; V(H)1, V(H)6, and V(H)11 were underutilized; and the majority of the V(H) segments belonged to the V(H)7, V(H)8, and V(H)9 families. Most of the nine J(H) segments seemed to be randomly used, except J(H)6 and J(H)9, which were found only once in 79 clones.
Asunto(s)
Ambystoma mexicanum/inmunología , Diversidad de Anticuerpos , Cadenas Pesadas de Inmunoglobulina/química , Región Variable de Inmunoglobulina/química , Familia de Multigenes/inmunología , Ambystoma mexicanum/genética , Secuencia de Aminoácidos , Animales , Diversidad de Anticuerpos/genética , Reordenamiento Génico de Linfocito B/inmunología , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Región de Unión de la Inmunoglobulina/química , Región de Unión de la Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Cadenas mu de Inmunoglobulina/química , Cadenas mu de Inmunoglobulina/genética , Ratones , Datos de Secuencia Molecular , Filogenia , XenopusRESUMEN
The immune capacity of young and adult axolotls (Ambystoma mexicanum) was evaluated by examining the combinatorial and junctional diversity of the VH chain. A large number of VDJ rearrangements isolated from 2.5-, 3.5-, 10-, and 24-month-old animals were sequenced. Six JH segments were identified with the canonical structure of all known vertebrate JHs, including the conserved Trp103-Gly104-X-Gly106 motif. Four core DH-like sequences were used by most (80%) of the VDJ junctions. These G-rich sequences had structures reminiscent of the TCRB DB sequences, and were equally used in their three reading frames. About 25% of the Igh, VDJ junctions from 3.5-month-old axolotls were out of frame, but most rearrangements were in frame at 10 and 24 months, suggesting that there is active selection of the productively rearranged Igh chains in the developing animals. There was no significant difference between the size of CDR3 in young (3.5 months) and subadult (10 months) axolotls (mean: 8.5 amino acids). However, the CDR3 loop was 1 amino acid longer in 2-year-old adult animals (mean: 9.5 residues). Several pairs of identical VDJ/CDR3 sequences were shared between 3.5-month-old individually analyzed axolotls, or between groups of axolotl of different ages. These identical rearrangements might be provided by the selection of some B-cell clones important for species survival, although the probability that different 3.5-month-old axolotl larvae would produce identical junctions seems very low, considering their limited number of B cells (less than 10(5)). The high frequency of tyrosine residues and the paucity of charged residues in the axolotl CDR3 loops may explain the polyreactivity of natural antibodies, and also clarify why it is so difficult to raise specific antibodies against soluble antigens.
Asunto(s)
Ambystoma/genética , Reordenamiento Génico , Cadenas Pesadas de Inmunoglobulina/genética , Región de Unión de la Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Ambystoma/crecimiento & desarrollo , Ambystoma/inmunología , Animales , Variación Genética , Cadenas mu de Inmunoglobulina/genética , Inmunoglobulinas/genética , Especificidad de la EspecieRESUMEN
The presence of multiple VHDJH joinings in upwards of 30% of acute lymphoblastic leukemias (ALL) suggests a relative instability of the rearranged immunoglobulin heavy chain (IgH) gene, but the mechanisms involved are not completely understood. An investigation of the structure of the VHDJH joinings using complementarity determining region (CDR)3 polymerase chain reaction (PCR) in 12 leukemias at both diagnosis and relapse indicates that this instability may increase as a function of time. In only one of seven cases in which relapse occurred within 3 years from diagnosis was a new VHDJH joining identified and this coexisted with the original diagnostic joining. Most strikingly, new VHDJH joinings were identified in four of five cases in which relapse occurred more than 5 years from diagnosis. In this latter population, the instability of the joinings was generated from VH----VH gene replacement events in two cases, since the new joinings retained the original DJH sequences and partial N region homology at the VHD junction, and probably in a third case from a VH gene rearrangement to a common DJH precursor. Furthermore, in five of 23 (21.7%) additional cases studied at diagnosis, subclones were identified that had similar modifications of the VH-N region. These data indicate that VH gene replacement events and VH gene rearrangements to a common DJH joining contribute to the instability of the VHDJH joining in ALL. This phenomenon should be taken into consideration in those methodologies that exploit IgH rearrangements for detection of minimal residual disease.
Asunto(s)
Linfoma de Burkitt/inmunología , Reordenamiento Génico , Genes de Inmunoglobulinas , Cadenas Pesadas de Inmunoglobulina/genética , Región de Unión de la Inmunoglobulina/genética , Adolescente , Secuencia de Bases , Linfoma de Burkitt/genética , Niño , Preescolar , ADN de Neoplasias/química , Humanos , Lactante , Datos de Secuencia Molecular , Recurrencia Local de Neoplasia/inmunología , Reacción en Cadena de la Polimerasa , Homología de Secuencia de Ácido NucleicoRESUMEN
Antigen receptors on most T-cells are heterodimeric glycoproteins, comprised of an alpha chain and a beta chain. These chains are encoded by discontiguous variable (V), diversity (D) and joining (J) gene segments that rearrange to produce a contiguous and functional alpha or beta chain gene. To investigate the size and diversity of the germline repertoire of alpha-chain gene segments, we have characterized and sequenced 20 alpha chain cDNAs. Among these cDNA clones, we have found 4 J alpha and 4 V alpha sequences that have not yet been described. The relationship of these "new" gene segments to those already characterized is discussed.