Babesia bovis Rad51 ortholog influences switching of ves genes but is not essential for segmental gene conversion in antigenic variation.

The tick-borne apicomplexan parasite, Babesia bovis, a highly persistent bovine pathogen, expresses VESA1 proteins on the infected erythrocyte surface to mediate cytoadhesion. The cytoadhesion ligand, VESA1, which protects the parasite from splenic passage, is itself protected from a host immune response by rapid antigenic variation. B. bovis relies upon segmental gene conversion (SGC) as a major mechanism to vary VESA1 structure. Gene conversion has been considered a form of homologous recombination (HR), a process for which Rad51 proteins are considered pivotal components. This could make BbRad51 a choice target for development of inhibitors that both interfere with parasite genome integrity and disrupt HR-dependent antigenic variation. Previously, we knocked out the Bbrad51 gene from the B. bovis haploid genome, resulting in a phenotype of sensitivity to methylmethane sulfonate (MMS) and apparent loss of HR-dependent integration of exogenous DNA. In a further characterization of BbRad51, we demonstrate here that ΔBbrad51 parasites are not more sensitive than wild-type to DNA damage induced by γ-irradiation, and repair their genome with similar kinetics. To assess the need for BbRad51 in SGC, RT-PCR was used to observe alterations to a highly variant region of ves1α transcripts over time. Mapping of these amplicons to the genome revealed a significant reduction of in situ transcriptional switching (isTS) among ves loci, but not cessation. By combining existing pipelines for analysis of the amplicons, we demonstrate that SGC continues unabated in ΔBbrad51 parasites, albeit at an overall reduced rate, and a reduction in SGC tract lengths was observed. By contrast, no differences were observed in the lengths of homologous sequences at which recombination occurred. These results indicate that, whereas BbRad51 is not essential to babesial antigenic variation, it influences epigenetic control of ves loci, and its absence significantly reduces successful variation. These results necessitate a reconsideration of the likely enzymatic mechanism(s) underlying SGC and suggest the existence of additional targets for development of small molecule inhibitors.

Diversification of the Primary Antibody Repertoire by AID-Mediated Gene Conversion.

Gene conversion, mediated by activation-induced cytidine deaminase (AID), has been found to contribute to generation of the primary antibody repertoire in several vertebrate species. Generation of the primary antibody repertoire by gene conversion of immunoglobulin (Ig) genes occurs primarily in gut-associated lymphoid tissues (GALT) and is best described in chicken and rabbit. Here, we discuss current knowledge of the mechanism of gene conversion as well as the contribution of the microbiota in promoting gene conversion of Ig genes. Finally, we propose that the antibody diversification strategy used in GALT species, such as chicken and rabbit, is conserved in a subset of human and mouse B cells.

Obtención de suspensiones celulares y embriones somáticos de cafeto (Coffea canephora P) con el empleo de metabolitos bacterianos / Obtaining of cell suspensions and somatic embryos of coffee (Coffea canephora P) using of bacterial metabolites

La embriogénesis somática es importante como sistema modelo para estudiar el desarrollo de eventos fisiológicos, citológicos y moleculares que sustentan la embriogénesis en plantas, por ser un sistema adecuado para la propagación masiva de especies vegetales y servir de herramienta para el mejoramiento genético, la conservación de germoplasma y la validación de nuevos productos biológicos, y facilitar la producción a gran escala a través del cultivo en medio líquido y su aplicación en biorreactores, proporcionando alta frecuencia de multiplicación, rápido crecimiento del embrión, facilidad de absorción de nutrientes y reducción de la labor de subcultivo. En este trabajo se empleó la embriogénesis somática como vía de multiplicación para evaluar el efecto de metabolitos bacterianos en la inducción de suspensiones celulares y embriones somáticos en tres genotipos de cafeto pertenecientes a Coffea canephora P. variedad Robusta. Para ello se estudiaron densidades de inóculo entre 0,2, 0,5, 1,0 y 3,0 gMF/L-1, y se evaluó el efecto de diferentes medios de cultivo en el desarrollo del proceso. Los resultados mostraron un comportamiento diferenciado en el genotipo M-28, en medios de cultivo suplementados con reguladores de crecimiento convencionales y en los alternativos. Se evidenció una fuerte relación entre la viabilidad celular y el número de células, ante las diferentes condiciones de cultivo y según la densidad de inóculo, se observó un amplio rango de tamaño y forma en las poblaciones de embriones somáticos. Los porcentajes de conversión de ES con el medio MDE-2 evidenciaron mejoras de este indicador para el cultivo del cafeto.

The somatic embryogenesis is important as model system to study the development of physiologic and molecular events that sustain the embryogenesis in plants, is an appropriate system for the massive propagation of vegetable species and as tool for the genetic improvement, the germplasm conservation and the validation of new biological products and to facilitate the multiplication to great scale through the culture in liquid medium, as well as application in bioreactores, providing high multiplication frequency, quick growth of the embryo, easiness of absorption of nutritious and reduction of the subculturing. In this paper the somatic embryogenesis was used to evaluate the effect of bacterial compounds in the induction of cellular suspensions and somatic embryos in three coffee genotypes of Coffea canephora P. var. Robusta. Were studied inoculo densities among 0.2, 0.5, 1.0 and 3.0 gMF/L-1 and the effect of different culture medium in the development of the process. The results showed a behavior differed in the genotype M-28, in medium culture with conventional regulators of growth and the alternatives. Strong relationship was evidenced between the cellular viability and the number of cells, in the different cultivation conditions and according to the inoculo density, a wide range of size and forms as observed in the populations of somatic embryos. The conversion percentages with the medium MDE-2, evidenced improvements of this indicator for the coffee.

Polymorphism of the HLA-B*15 group of alleles is generated following 5 lineages of evolution.

Generation of the HLA-B*15 group of alleles has been analyzed using exon 1, intron 1, exon 2, intron 2, and exon 3 sequences from human and nonhuman primates. Results indicated that the 230 alleles analyzed could be grouped into 5 different lineages of evolution coming from nonhuman primate MHC-B* alleles sharing characteristic nucleotide sequences. The major evolutionary mechanism of evolution in this group of alleles is the gene conversion event with the exchange of genomic sequences present in other HLA-B*alleles. This evolutionary event reflects the importance of the exchanges between different genomic regions of distinct HLA-A*, -B*, or -C* alleles and only 1 group of HLA-B* alleles (B*15 in the present paper). These data also correlated with the geographic distribution of the lineages postulated and with the corresponding serologic specificities (B62, -63, -71, -72, -75, -76, and -77). In conclusion, the high degree of polymorphism of 1 group of alleles has a specific and simple pathway of evolution, which could result in new insight into the study of immune system functionality, disease association studies, and anthropological studies.

Cutting edge: a cis-acting DNA element targets AID-mediated sequence diversification to the chicken Ig light chain gene locus.

Somatic hypermutation and gene conversion are two closely related processes that increase the diversity of the primary Ig repertoire. Both processes are initiated by the activation-induced cytidine deaminase that converts cytosine residues to uracils in a transcription-dependent manner; these lesions are subsequently fixed in the genome by direct replication and error-prone DNA repair. Two alternative mechanisms were proposed to explain why this mutagenic activity is targeted almost exclusively to Ig loci: 1) specific cis-acting DNA sequences; or 2) very high levels of Ig gene transcription. In this study we now identify a novel 3' regulatory region in the chicken Ig light chain gene containing not only a classical transcriptional enhancer but also cis-acting DNA elements essential for targeting activation-induced cytidine deaminase-mediated sequence diversification to this locus.

Role of activation-induced deaminase protein kinase A phosphorylation sites in Ig gene conversion and somatic hypermutation.

Activation-induced deaminase (AID) is thought to initiate somatic hypermutation (SHM), gene conversion (GCV), and class switch recombination (CSR) by the transcription-coupled deamination of cytosine residues in Ig genes. Phosphorylation of AID by protein kinase A (PKA) and subsequent interaction of AID with replication protein A (RPA) have been proposed to play important roles in allowing AID to deaminate DNA during transcription. Serine 38 (S38) of mouse AID is phosphorylated in vivo and lies in a consensus target site for PKA, and mutation of this residue interferes with CSR and SHM. In this study, we demonstrate that S38 in mouse and chicken AID is phosphorylated in chicken DT40 cells and is required for efficient GCV and SHM in these cells. Paradoxically, zebra fish AID, which lacks a serine at the position corresponding to S38, has previously been shown to be active for CSR and we demonstrate that it is active for GCV/SHM. Aspartate 44 (D44) of zebra fish AID has been proposed to compensate for the absence of the S38 phosphorylation site but we demonstrate that mutation of D44 has no effect on GCV/SHM. Some features of zebra fish AID other than D44 might compensate for the absence of S38. Alternatively, the zebra fish protein might function in a manner that is independent of PKA and RPA in DT40 cells, raising the possibility that, under some circumstances, AID mediates efficient Ig gene diversification without the assistance of RPA.

Maintenance of antibody to pathogen epitopes generated by segmental gene conversion is highly dynamic during long-term persistent infection.

Multiple bacterial and protozoal pathogens utilize gene conversion to generate rapid intrahost antigenic variation. Both large- and small-genome pathogens expand the size of the variant pool via a combinatorial process in which oligonucleotide segments from distinct donor loci are recombined in various combinations into expression sites. Although the potential combinatorial diversity generated by this segmental gene conversion mechanism is quite large, the functional variant pool depends on whether immune responses against the recombined segments are generated and maintained, regardless of their specific combinatorial context. This question was addressed by tracking the Anaplasma marginale variant population and corresponding segment-specific immunoglobulin G (IgG) antibody responses during long-term infection. Antibody was induced early in A. marginale infection, predominately against the surface-exposed hypervariable region (HVR) rather than against the invariant conserved flanking domains, and these HVR oligopeptides were most immunogenic at the time of acute bacteremia, when the variant population is derived via recombination from a single donor locus. However antibody to HVR oligopeptides was not consistently maintained during persistent infection, despite reexpression of the same segment, although in a different combinatorial context. This dynamic antibody recognition over time was not attributable to the major histocompatibility complex haplotype of individual animals or use of specific msp2 donor alleles. In contrast, the position and context of an individual oligopeptide segment within the HVR were significant determinants of antibody recognition. The results unify the genetic potential of segmental gene conversion with escape from antibody recognition and identify immunological effects of variant mosaic structure.

Elucidation of exon 1, 4, and 5 sequences of 39 infrequent HLA-B alleles.

More than 590 human leukocyte antigen (HLA)-B alleles have been identified by sequence analysis. Although the polymorphic exon 2 and 3 sequences of all HLA-B alleles are described, the sequences of the other exons of a number of infrequent B-alleles are unknown. In this study, the exon 1, 4, and 5 sequences of 39 different HLA-B alleles were elucidated by allele-specific sequencing. Overall, these exon sequences showed identity with the majority of the known sequences from the corresponding allele groups, except for four alleles B*4010, B*4415, B*4416, and B*5606. The exon 1 sequence of B*4010 had nucleotide differences with all B*40 alleles, but was identical to the B*54, *55, *56, and *59 allele groups. B*4416 differed from B*440201 at position 988, which was previously considered a conserved position. B*4415 showed exon 1, 4, and 5 sequences deviating from the other B*44 alleles, but identical to B*4501. The exon 1 and 4 sequences of B*5606 differed from other B*56 alleles, but were in complete agreement with B*7801. The deviating exon sequences of B*4415 and B*5606 confirmed the evolutionary origin of these alleles suggested by the sequences of exons 2 and 3. The polymorphism observed in exons 1, 4, and 5 merely reflects the lineage-specificity of HLA-B.

The MRE11-RAD50-NBS1 complex accelerates somatic hypermutation and gene conversion of immunoglobulin variable regions.

Targeted diversification of immunoglobulin variable regions is induced by activation-induced deaminase and may occur by either somatic hypermutation or gene conversion. MRE11-RAD50-NBS1 (MRN) is a ubiquitous and conserved nuclease complex critical for DNA break repair and is essential in class-switch recombination. Here we show that ectopic expression of NBS1, the regulatory subunit of MRN, accelerated hypermutation in the human B cell line Ramos and accelerated gene conversion in the chicken B cell line DT40. In both cases, accelerated diversification depended on MRN complex formation. These data suggest that MRN promotes DNA cleavage and/or mutagenic repair of lesions initiated by activation-induced deaminase, acting in the shared pathway of immunoglobulin gene diversification.

Analysis of mutational lineage trees from sites of primary and secondary Ig gene diversification in rabbits and chickens.

Lineage trees of mutated rearranged Ig V region sequences in B lymphocyte clones often serve to qualitatively illustrate claims concerning the dynamics of affinity maturation. In this study, we use a novel method for analyzing lineage tree shapes, using terms from graph theory to quantify the differences between primary and secondary diversification in rabbits and chickens. In these species, Ig gene diversification starts with rearrangement of a single (in chicken) or a few (in rabbit) V(H) genes. Somatic hypermutation and gene conversion contribute to primary diversification in appendix of young rabbits or in bursa of Fabricius of embryonic and young chickens and to secondary diversification during immune responses in germinal centers (GCs). We find that, at least in rabbits, primary diversification appears to occur at a constant rate in the appendix, and the type of Ag-specific selection seen in splenic GCs is absent. This supports the view that a primary repertoire is being generated within the expanding clonally related B cells in appendix of young rabbits and emphasizes the important role that gut-associated lymphoid tissues may play in early development of mammalian immune repertoires. Additionally, the data indicate a higher rate of hypermutation in rabbit and chicken GCs, such that the balance between hypermutation and selection tends more toward mutation and less toward selection in rabbit and chicken compared with murine GCs.

Immunoglobulin gene conversion in chicken DT40 cells largely proceeds through an abasic site intermediate generated by excision of the uracil produced by AID-mediated deoxycytidine deamination.

Diversification of the primary antibody repertoire in chickens is achieved by a gene conversion process that uses a set of immunoglobulin variable (IgV) pseudogenes as templates. Studies usingthe chicken DT40 B lymphoma cell line have shown that this gene conversion is dependent on activation-induced deaminase, which deaminates deoxycytidine to deoxyuridine in the IgV gene. The mechanism by which the resultant deoxyuridine/deoxyguanosine (dU/dG) mismatch acts to initiate the gene conversion process is unknown but likely involves either (i) recognition of the dU/dG pair by the mismatch repair complex or (ii) recognition of the dU itself by uracil-DNA glycosylase. To discriminate these possibilities, we have investigated the effects on IgV gene conversion of inhibiting uracil-DNA glycosylase. We find that such inhibition diminishes gene conversion, biasing instead towards point mutations. These results demonstrate that IgV gene conversion in DT40 cells is substantially dependent on uracil excision and implies that it proceeds by a pathway involving an abasic site, which could be acted upon by an apyrimidinic endonuclease to generate a DNA strand break facilitating the conversion process.

Allelic variation at the VHa locus in natural populations of rabbit (Oryctolagus cuniculus, L.).

The large interallelic distances between the three rabbit Ig V(H)a lineages, a1, a2 and a3, suggest that the persistence time of the V(H)a polymorphism could amount to 50 million years, which is much longer than that of MHC polymorphisms. Rabbit originated in the Iberian Peninsula where two subspecies coexist, one of which is confined to Southwestern Iberia (Oryctolagus cuniculus algirus). We studied the V(H) loci in the original species range to obtain a better understanding of the evolutionary history of this unusual polymorphism. Serological surveys revealed that sera from the subspecies algirus, when tested with V(H)a locus-specific alloantisera, showed either cross-reactivity ("a-positive" variants) or no reaction at all ("a-blank"). Using RT-PCR, we determined 120 sequences of rearranged V(H) genes expressed in seven algirus rabbits that were typed as either a-positive or a-blank. The data show that the V(H) genes transcribed in a-positive rabbits are closely related to the V(H)1 alleles of domestic rabbits. In contrast, a-blank rabbits were found to preferentially use V(H) genes that, although clearly related to the known V(H)a genes, define a new major allotypic lineage, designated a4. The a4 sequences have hallmark rabbit V(H)a residues together with a number of unprecedented amino acid changes in framework region 2 and 3. The net protein distances between the V(H)a4 and the V(H)a1, a2, and a3 lineages were 20, 29, and 21% respectively. We conclude that at least four distantly related lineages of the rabbit V(H)a locus exist, one of which seems to be endemic in the Iberian range.

Gene conversion-like sequence transfers between transgenic antibody V genes are independent of RAD54.

Homology-based Ig gene conversion is a major mechanism for Ab diversification in chickens and the Rad54 DNA repair protein plays an important role in this process. In mice, although gene conversion appears to be rare among endogenous Ig genes, Ab H chain transgenes undergo isotype switching and gene conversion-like sequence transfer processes that also appear to involve homologous recombination or gene conversion. Furthermore, homology-based DNA repair has been suggested to be important for somatic mutation of endogenous mouse Ig genes. To assess the role of Rad54 in these mouse B cell processes, we have analyzed H chain transgene isotype switching, sequence transfer, and somatic hypermutation in mice that lack RAD54. We find that Rad54 is not required for either transgene switching or transgene hypermutation. Furthermore, even transgene sequence transfers that are known to require homology-based recombinations are Rad54 independent. These results indicate that mouse B cells must use factors for promoting homologous recombination that are distinct from the Rad54 proteins important in homology-based chicken Ab gene recombinations. Our findings also suggest that mouse H chain transgene sequence transfers might be more closely related to an error-prone homology-based somatic hypermutational mechanism than to the hyperconversion mechanism that operates in chicken B cells.

Twelve novel HLA-B*15 alleles carrying previously observed sequence motifs are placed into B*15 subgroups.

Twelve new B*15 alleles are described. All of the known B*15 alleles are divided into subgroups based on serologic assignments and/or nucleotide sequence polymorphisms. These groups might be used as a reference for DNA-based testing at an intermediate (i.e. "serologic") level of resolution.

Novel HLA allele, HLA-B*4013, identified from a potential bone marrow recipient. Putative gene conversion events with donor sequence.

A new B40 allele was identified in a leukemic Caucasian patient. This allele, designated B*4013, differs in alpha 1 domain from B*4002 at six amino acidic positions: 67, 77, 80, 81, 82 and 83. Most of this substitutions could alter the antigen binding site of the HLA-B molecule. B*4013 may have originated by gene conversion or reciprocal recombination involving B*4002 as the recipient allele of sequence donated by B*4406. The new allele was serologically typed as a "blank" associated with the Bw4 epitope.

Identification of a new DQB1 allele that appears to have been generated by an interallelic sequence exchange.

The study reports the molecular characterization of a new DQB1 variant initially detected by unusual sequence-specific oligonucleotide (SSO) hybridization patterns in one Caucasoid individual. This new allele is identical to DQB1*0501 except for two silent nucleotide substitutions at codons 49 (GCA-->GCG) and 77 (AGG-->AGA). Compared with DQB1*0502 it differs in three nucleotides at codon 57 changing AGC (encoding Ser) to GTT (encoding Val). Considering the paternal genotype, it appears this new allele might have been generated by an interallelic sequence exchange between the two paternal DQB1 alleles.

Gene-conversion in rabbit B-cell ontogeny and during immune responses in splenic germinal centers.

Combinatorial diversity is limited in rabbits because only a few V(H) genes rearrange. Most diversification of the primary repertoire is generated by somatic hypermutation and gene conversion-like changes of rearranged V(H) in B cells that migrate to appendix and other gut associated lymphoid tissues (GALT) of young rabbits. The changes are referred to as gene conversion-like because the non-reciprocal nature of the alterations introduced has not yet been demonstrated. There are many similarities between rabbits and chickens in how their B cells develop and diversify their repertoires. However, although the majority of rabbit B cells may have rearranged and diversified their V genes early in life, some B cells in adult rabbits have rearranged VH sequences that are identical or nearly identical to germline sequences. We found these cells in splenic germinal centers (GC) on days 7 and 10 after immunization of normal adult rabbits with DNP-BGG. By day 15, all rearranged V(H) sequences were diversified. We find an overall pattern of splenic precursor cells whose germline or near germline sequences change both by gene conversion and point mutations during early divisions and mainly by point mutations during later divisions. These events, in parallel with diversification of light chain sequences, may produce the diverse combining sites that serve as substrates for further affinity maturation by selection either within GC or later among emigrant cells in sites such as bone marrow. Some of the sequences altered by gene conversion in splenic germinal centers may also produce new members of the B-cell repertoire in adult rabbits comparable to those produced in GALT of neonatal rabbits.

Gene conversion events contribute to the polymorphic variation of the surrogate light chain gene lambda 5/14.1.

Normally occurring and experimentally induced models of immunodeficiency indicate that B cell development and antibody production are influenced by genetic factors. It is highly likely that polymorphic variants in genes that encode receptors for growth and differentiation factors, signal transduction molecules, and components of the B cell and pre-B-cell receptor complex contribute to this genetic control. We have identified a surprisingly large number of polymorphic variants in lambda5/14.1. Together with VpreB, lambda5/14.1 forms the surrogate light chain in the pre-B-cell receptor complex. Thirteen variant alleles of lambda5/14.1 were found in 134 unrelated individuals. Nine of these variants result in changes in the amino acid sequence of this small protein. The majority of the single base pair substitutions in lambda5/14.1 could be attributed to gene conversion events in which donor sequences from the lambda5 pseudogenes, 16.1, 16.2, and Glambda1, replace the wild-type sequence in the lambda5/14.1 functional gene. These findings indicate that gene conversion events play a major role in generating diversity that could affect stability or expression of the pre-B-cell receptor complex.

Antigen-induced somatic diversification of rabbit IgH genes: gene conversion and point mutation.

During T cell-dependent immune responses in mouse and human, Ig genes diversify by somatic hypermutation within germinal centers. Rabbits, in addition to using somatic hypermutation to diversify their IgH genes, use a somatic gene conversion-like mechanism, which involves homologous recombination between upstream VH gene segments and the rearranged VDJ genes. Somatic gene conversion and somatic hypermutation occur in young rabbit gut-associated lymphoid tissue and are thought to diversify a primary Ab repertoire that is otherwise limited by preferential VH gene segment utilization. Because somatic gene conversion is rarely found within Ig genes during immune responses in mouse and human, we investigated whether gene conversion in rabbit also occurs during specific immune responses, in a location other than gut-associated lymphoid tissue. We analyzed clonally related VDJ genes from popliteal lymph node B cells responding to primary, secondary, and tertiary immunization with the hapten FITC coupled to a protein carrier. Clonally related VDJ gene sequences were derived from FITC-specific hybridomas, as well as from Ag-induced germinal centers of the popliteal lymph node. By analyzing the nature of mutations within these clonally related VDJ gene sequences, we found evidence not only of ongoing somatic hypermutation, but also of ongoing somatic gene conversion. Thus in rabbit, both somatic gene conversion and somatic hypermutation occur during the course of an immune response.