Single-Cell Sequencing Confirms Transcripts and VHDJH Rearrangements of Immunoglobulin Genes in Human Podocytes.

Most glomerular diseases are associated with inflammation caused by deposited pathogenic immunoglobulins (Igs), which are believed to be produced by B cells. However, our previous study indicated that the human podocyte cell line can produce IgG. In this study, we aimed to confirm the transcripts and characterize the repertoires of Igs in primary podocytes at single cell level. First, single-cell RNA sequencing of cell suspensions from "normal" kidney cortexes by a 10xGenomics Chromium system detected Ig transcripts in 7/360 podocytes and Ig gene segments in 106/360 podocytes. Then, we combined nested PCR with Sanger sequencing to detect the transcripts and characterize the repertoires of Igs in 48 single podocytes and found that five classes of Ig heavy chains were amplified in podocytes. Four-hundred and twenty-nine VHDJH rearrangement sequences were analyzed; podocyte-derived Igs exhibited classic VHDJH rearrangements with nucleotide additions and somatic hypermutations, biased VH1 usage and restricted diversity. Moreover, compared with the podocytes from healthy control that usually expressed one class of Ig and one VHDJH pattern, podocytes from patients expressed more classes of Ig, VHDJH patterns and somatic hypermutations. These findings suggested that podocytes can express Igs in normal condition and increase diversity in pathological situations.

Multiple IgH Isotypes Including IgD, Subclasses of IgM, and IgY Are Expressed in the Common Ancestors of Modern Birds.

Although evolutionarily just as ancient as IgM, it has been thought for many years that IgD is not present in birds. Based on the recently sequenced genomes of 48 bird species as well as high-throughput transcriptome sequencing of immune-related tissues, we demonstrate in this work that the ostrich (Struthio camelus) possesses a functional δ gene that encodes a membrane-bound IgD H chain with seven CH domains. Furthermore, δ sequences were clearly identified in many other bird species, demonstrating that the δ gene is widely distributed among birds and is only absent in certain bird species. We also show that the ostrich possesses two µ genes (µ1, µ2) and two υ genes (υ1, υ2), in addition to the δ and α genes. Phylogenetic analyses suggest that subclass diversification of both the µ and υ genes occurred during the early stages of bird evolution, after their divergence from nonavian reptiles. Although the positions of the two υ genes are unknown, physical mapping showed that the remaining genes are organized in the order µ1-δ-α-µ2, with the α gene being inverted relative to the others. Together with previous studies, our data suggest that birds and nonavian reptile species most likely shared a common ancestral IgH gene locus containing a δ gene and an inverted α gene. The δ gene was then evolutionarily lost in selected birds, whereas the α gene lost in selected nonavian reptiles. The data obtained in this study provide significant insights into the understanding of IgH gene evolution in tetrapods.

Comparative study of IgA VH 3 gene usage in healthy TST(-) and TST(+) population exposed to tuberculosis: deep sequencing analysis.

The acquired immune response against tuberculosis is commonly associated with T-cell responses with little known about the role of B cells or antibodies. There have been suggestions that B cells and humoral immunity can modulate the immune response to Mycobacterium tuberculosis. However, the mechanisms involving B-cell responses in M. tuberculosis are not fully understood, in particular the antibody gene preferences. We hypothesized that a preferential use of V genes can be seen associated with resistance to infection mainly in the IgA isotype, which is of prominent importance for infection by pathogens via the mucosal route. We studied healthy individuals with long-term exposure to tuberculosis, infected (TST(+) ) and uninfected TST(-) ) with M. tuberculosis. From a total of 22 V genes analysed, the TST(-) population preferred the VH 3-23 and Vκ1 genes. The VH 3-23 genes were subsequently subjected to 454 amplicon sequencing. The TST(-) population showed a higher frequency of the D3-10 segment compared with the D3-22 segment for the TST(+) population. The J segment usage pattern was similar for both populations with J4 segment being used the most. A preferential pairing of J4 segments to D3-3 was seen for the TST(-) population. The antibodyome difference between both populations suggests a preference for antibodies with VH 3-23, D3-3, JH 4 gene usage by the TST(-) population that could be associated with resistance to infection with M. tuberculosis.

The ataxia telangiectasia mutated and cyclin D3 proteins cooperate to help enforce TCRß and IgH allelic exclusion.

Coordination of V rearrangements between loci on homologous chromosomes is critical for Ig and TCR allelic exclusion. The Ataxia Telangietasia mutated (ATM) protein kinase promotes DNA repair and activates checkpoints to suppress aberrant Ig and TCR rearrangements. In response to RAG cleavage of Igκ loci, ATM inhibits RAG expression and suppresses further Vκ-to-Jκ rearrangements to enforce Igκ allelic exclusion. Because V recombination between alleles is more strictly regulated for TCRß and IgH loci, we evaluated the ability of ATM to restrict biallelic expression and V-to-DJ recombination of TCRß and IgH genes. We detected greater frequencies of lymphocytes with biallelic expression or aberrant V-to-DJ rearrangement of TCRß or IgH loci in mice lacking ATM. A preassembled DJß complex that decreases the number of TCRß rearrangements needed for a productive TCRß gene further increased frequencies of ATM-deficient cells with biallelic TCRß expression. IgH and TCRß proteins drive proliferation of prolymphocytes through cyclin D3 (Ccnd3), which also inhibits VH transcription. We show that inactivation of Ccnd3 leads to increased frequencies of lymphocytes with biallelic expression of IgH or TCRß genes. We also show that Ccnd3 inactivation cooperates with ATM deficiency to increase the frequencies of cells with biallelic TCRß or IgH expression while decreasing the frequency of ATM-deficient lymphocytes with aberrant V-to-DJ recombination. Our data demonstrate that core components of the DNA damage response and cell cycle machinery cooperate to help enforce IgH and TCRß allelic exclusion and indicate that control of V-to-DJ rearrangements between alleles is important to maintain genomic stability.

The role of BCR isotype in B-cell development and activation.

The development and function of B lymphocytes critically depend on the non-germline B-cell antigen receptor (BCR). In addition to the diverse antigen-recognition regions, whose coding sequences are generated by the somatic DNA rearrangement, the variety of the constant domains of the Heavy Chain (HC) portion contributes to the multiplicity of the BCR types. The functions of particular classes of the HC, particularly in the context of the membrane BCR, are not completely understood. The expression of the various classes of the HC correlates with the distinct stages of B-cell development, types of B-cell subsets, and their effector functions. In this chapter, we summarize and discuss the accumulated knowledge on the role of the µ, δ, and γ HC isotypes of the conventional and precursor BCR in B-cell differentiation, selection, and engagement with (auto)antigens.

Manipulating mIgD-expressing B cells with anti-migis-δ monoclonal antibodies.

Surface IgD and IgM doubly positive cells comprise the major population of B cells in the human immune system. The heavy chain of membrane-bound IgD (mδ) differs from that of IgD (δ) in that mδ contains a C-terminal membrane-anchor peptide. Our group previously proposed that the N-terminal extracellular segment of 27 aa residues of the membrane-anchor peptide of mδ, referred to as the mIg isotype-specific-δ (migis-δ) segment, may provide a unique antigenic site for isotype-specific targeting of mIgD(+) B cells. Here we report the preparation of mouse mAbs specific for human migis-δ. The mAbs bound to human migis-δ-containing recombinant proteins in an ELISA and to mIgD-expressing transfectants of a CHO cell line as analyzed by flow cytometry. MAb 20E6, which binds to an epitope toward the N-terminal of human migis-δ, could stain human B cell line MC116, which expressed mIgD and mIgM. MC116 cells could be induced to undergo apoptosis by treatment with 20E6 in the presence of a second crosslinking antibody. Chimeric 20E6 caused antibody-dependent cellular cytotoxicity of MC116 cells in the presence of human PBMCs as the source of effector cells. In cultures of PBMCs, 20E6 down-regulated the population of mIgD(+) B cells. The production of human IgM by transplanted MC116 cells in NOD-SCID (NOD.CB17-Prkdc(scid)/IcrCrlBltw) mice could be suppressed by 20E6. These results encourage further investigation of the potential of anti-migis-δ mAbs to control mIgD(+) B cells, when such a manipulation may alleviate a disease state.

Extensive diversification of IgD-, IgY-, and truncated IgY(δFc)-encoding genes in the red-eared turtle (Trachemys scripta elegans).

IgY(ΔFc), containing only CH1 and CH2 domains, is expressed in the serum of some birds and reptiles, such as ducks and turtles. The duck IgY(ΔFc) is produced by the same υ gene that expresses the intact IgY form (CH1-4) using different transcriptional termination sites. In this study, we show that intact IgY and IgY(ΔFc) are encoded by distinct genes in the red-eared turtle (Trachemys scripta elegans). At least eight IgY and five IgY(ΔFc) transcripts were found in a single turtle. Together with Southern blotting, our data suggest that multiple genes encoding both IgY forms are present in the turtle genome. Both of the IgY forms were detected in the serum using rabbit polyclonal Abs. In addition, we show that multiple copies of the turtle δ gene are present in the genome and that alternative splicing is extensively involved in the generation of both the secretory and membrane-bound forms of the IgD H chain transcripts. Although a single µ gene was identified, the α gene was not identified in this species.

Identification of IGHCδ-BACH2 fusion transcripts resulting from cryptic chromosomal rearrangements of 14q32 with 6q15 in aggressive B-cell lymphoma/leukemia.

In B-cell malignancies, genes implicated in B-cell differentiation, germinal center formation, apoptosis, and cell cycle regulation are juxtaposed to immunoglobulin loci through chromosomal translocations. In this study, we identified the BTB and CNC homology 2 (BACH2) gene as a novel translocation partner of the immunoglobulin heavy chain (IGH) locus in a patient with IGH-MYC-positive, highly aggressive B-cell lymphoma/leukemia carrying der(14)t(8;14) and del(6)(q15). Fluorescence in situ hybridization analysis using an IGH/MYC probe detected an IGH-MYC fusion signal on der(14) and IGH signal on del(6). Genome copy number analysis showed a deletion in the 6q15-25 region and a centromeric breakpoint within the BACH2 gene. cDNA bubble polymerase chain reaction using BACH2 primers revealed that the first exon of Cδ was fused to the 5'-untranslated region of BACH2 exon 2. The Cδ-BACH2 fusion transcript consisted of exon 1 of Cδ and exons 2 to 9 of BACH2, encompassing the entire BACH2 coding region, and the BACH2 was highly expressed in this patient. These results indicate that Cδ-BACH2 fusion may cause constitutive activation of BACH2. Although additional screening of 47 samples of B-cell non-Hodgkin's lymphoma (B-NHL) patients and 29 cell lines derived from B-cell malignancies by double-color fluorescence in situ hybridization analysis detected a split signal with deletion of centromeric region of BACH2 only in a patient with follicular lymphoma, BACH2 was highly expressed in lymphoma cells of the patient and B-NHL cell lines with IGH-MYC translocation. These findings suggest that BACH2 plays a critical role in B-cell lymphomagenesis, especially related to IGH-MYC translocation in some way.

Molecular cloning of IgT from Atlantic salmon, and analysis of the relative expression of τ, µ, and δ in different tissues.

In the present study, IgT genes of Atlantic salmon were cloned and characterised. Analysis of our sequence data as well as ESTs reported to the databases revealed three distinct IgT heavy chain sub-variants in salmon, as opposed to two of IgM and IgD. The IgT sub-variants in salmon are 76-80% identical to each other, and 75-82% identical to the reported rainbow trout sequences, whereas the similarity to the orthologous molecules in zebrafish, grass carp, mandarin fish, and grouper is 25-41%. The heavy chains of both secreted and membrane anchored forms of salmon IgT include four constant Ig domains, τ1-τ4. This parallels the IgM heavy chains in elasmobranch fish and higher vertebrates, but differs from IgM in teleost fish where the membrane anchored form include only three constant Ig domains, µ1-µ3. The similarity between τ1 and µ1 in salmon is relatively high (52%) when compared to the remaining part of the molecules (τ2-τ4 and µ2-µ4 are 13-24% similar). To compare τ, µ and δ expressions in different tissues (head kidney, thymus, spleen, gill, skin, hind gut, brain and muscle) of Atlantic salmon, RT-qPCR assays were designed and evaluated. The analyses revealed that IgM transcripts are most abundant (up to 200 times more than IgD) followed by IgT (up to 20 times more than IgD) in most tissues. Highest expression of IgM, IgT, and IgD was in head kidney and spleen.

Ornithorhynchus anatinus (platypus) links the evolution of immunoglobulin genes in eutherian mammals and nonmammalian tetrapods.

The evolutionary origins of mammalian immunoglobulin H chain isotypes (IgM, IgD, IgG, IgE, and IgA) are still incompletely understood as these isotypes differ considerably in structure and number from their counterparts in nonmammalian tetrapods. We report in this study that the platypus (Ornithorhynchus anatinus) Ig H chain constant region gene locus contains eight Ig encoding genes, which are arranged in an mu-delta-omicron-gamma2-gamma1-alpha1-epsilon-alpha2 order, spanning a total of approximately 200 kb DNA, encoding six distinct isotypes. The omicron (omicron for Ornithorhynchus) gene encodes a novel Ig H chain isotype that consists of four constant region domains and a hinge, and is structurally different from any of the five known mammalian Ig classes. This gene is phylogenetically related to upsilon (epsilon) and gamma, and thus appears to be a structural intermediate between these two genes. The platypus delta gene encodes ten heavy chain constant region domains, lacks a hinge region and is similar to IgD in amphibians and fish, but strikingly different from that in eutherian mammals. The platypus Ig H chain isotype repertoire thus shows a unique combination of genes that share similarity both to those of nonmammalian tetrapods and eutherian animals and demonstrates how phylogenetically informative species can be used to reconstruct the evolutionary history of functionally important genes.

Use of IGHJ and IGHD gene mutations in analysis of immunoglobulin sequences for the prognosis of chronic lymphocytic leukemia.

The level of somatic point mutation in immunoglobulin genes is an important prognostic indicator for patients with chronic lymphocytic leukemia (CLL). Mutation analysis presently focuses solely upon the heavy chain IGHV gene, however mutation is a stochastic process that also targets IGHD and IGHJ genes. Here, we evaluate the completeness and reliability of the reported IGHJ gene repertoire, and demonstrate the likely consequences of the inclusion of IGHD and IGHJ mutations in CLL analysis, using a dataset of 607 sequences. Inclusion of these mutations would lead to the re-classification of many sequences, which should significantly improve the prognostic value of mutation analysis.

IgD, like IgM, is a primordial immunoglobulin class perpetuated in most jawed vertebrates.

IgD has remained a mysterious Ig class and a bane to immunology students since its discovery >40 years ago. Its spotty occurrence in mammals and birds and the discovery of an isotype with similarities to IgD in bony fish are perplexing. We have identified IgD heavy (H) chain (delta) from the amphibian Xenopus tropicalis during examination of the IgH locus. The Xenopus delta gene is in the same position, immediately 3' of the IgM gene, as in mammals, and it is expressed only in the spleen at low levels, primarily as a transmembrane receptor by surface IgM(+) cells. Our data suggest that frog IgD is expressed on mature B cells, like in mouse/human. Unexpectedly, Xenopus IgD is orthologous to IgW, an Ig isotype found only in cartilaginous fish and lungfish, demonstrating that IgD/W, like IgM, was present in the ancestor of all living jawed vertebrates. In striking contrast to IgM, IgD/W is evolutionarily labile, showing many duplications/deletions of domains, the presence of multiple splice forms, existence as predominantly a secretory or transmembrane form, or loss of the entire gene in a species-specific manner. Our study suggests that IgD/W has played varied roles in different vertebrate taxa since the inception of the adaptive immune system, and it may have been preserved as a flexible locus over evolutionary time to complement steadfast IgM.

Molecular characterization of immunoglobulin D in mammals: immunoglobulin heavy constant delta genes in dogs, chimpanzees and four old world monkey species.

Antibodies are adaptor molecules that neutralize pathogens and link humoral and cellular defence mechanisms. Immunoglobulin D (IgD), one of the five antibody classes present in mammals, is expressed as an antigen receptor on naïve B cells. The functional role that IgD plays in the immune response is still poorly understood, but the recent characterization of immunoglobulin heavy constant delta genes (IGHD) in a variety of species challenges the view that IgD is of minor importance and is not present in many animals. On the basis of serological studies, IgD appears to be expressed in the majority of mammalian species examined. To confirm, at the molecular level, that IgD is present in different species, we cloned and sequenced IGHD cDNA from dogs and five non-human primate species (chimpanzee, rhesus macaque, cynomolgus macaque, baboon and sooty mangabey). Our results show that in all six species, IgD heavy chains possess three immunoglobulin domains and a long hinge region encoded by two exons. Only the hinge region of non-human primates is similar to the human hinge region, with conservation of O-glycosylation sites and multiple charged residues at opposing ends. The preservation of IgD in primates, dogs and previously characterized species suggests an important functional role for IgD, possibly involving binding to a receptor. The high degree of similarity existing between the structural features of human and non-human primate IgD suggests that non-human primates are suitable for in vivo studies designed to define the role that IgD plays in the immune response.

Reconsidering the human immunoglobulin heavy-chain locus: 1. An evaluation of the expressed human IGHD gene repertoire.

We have used a bioinformatics approach to evaluate the completeness and functionality of the reported human immunoglobulin heavy-chain IGHD gene repertoire. Using the hidden Markov-model-based iHMMune-align program, 1,080 relatively unmutated heavy-chain sequences were aligned against the reported repertoire. These alignments were compared with alignments to 1,639 more highly mutated sequences. Comparisons of the frequencies of gene utilization in the two databases, and analysis of features of aligned IGHD gene segments, including their length, the frequency with which they appear to mutate, and the frequency with which specific mutations were seen, were used to determine the reliability of alignments to the less commonly seen IGHD genes. Analysis demonstrates that IGHD4-23 and IGHD5-24, which have been reported to be open reading frames of uncertain functionality, are represented in the expressed gene repertoire; however, the functionality of IGHD6-25 must be questioned. Sequence similarities make the unequivocal identification of members of the IGHD1 gene family problematic, although all genes except IGHD1-14*01 appear to be functional. On the other hand, reported allelic variants of IGHD2-2 and of the IGHD3 gene family appear to be nonfunctional, very rare, or nonexistent. Analysis also suggests that the reported repertoire is relatively complete, although one new putative polymorphism (IGHD3-10*p03) was identified. This study therefore confirms a surprising lack of diversity in the available IGHD gene repertoire, and restriction of the germline sequence databases to the functional set described here will substantially improve the accuracy of IGHD gene alignments and therefore the accuracy of analysis of the V-D-J junction.

The immunoglobulin heavy-chain locus in zebrafish: identification and expression of a previously unknown isotype, immunoglobulin Z.

The only immunoglobulin heavy-chain classes known so far in teleosts have been mu and delta. We identify here a previously unknown class, immunoglobulin zeta, expressed in zebrafish and other teleosts. In the zebrafish heavy-chain locus, variable (V) gene segments lie upstream of two tandem diversity, joining and constant (DJC) clusters, resembling the mouse T cell receptor alpha (Tcra) and delta (Tcrd) locus. V genes rearrange to (DJC)(zeta) or to (DJC)(mu) without evidence of switch rearrangement. The zebrafish immunoglobulin zeta gene (ighz) and mouse Tcrd, which are proximal to the V gene array, are expressed earlier in development. In adults, ighz was expressed only in kidney and thymus, which are primary lymphoid organs in teleosts. This additional class adds complexity to the immunoglobulin repertoire and raises questions concerning the evolution of immunoglobulins and the regulation of the differential expression of ighz and ighm.

Human immunoglobulin selection associated with class switch and possible tolerogenic origins for C delta class-switched B cells.

Current paradigms of peripheral B cell selection suggest that autoreactive B cells are controlled by clonal deletion, anergy, and developmental arrest. We report that changes to the human antibody repertoire likely resulting from these mechanisms both for a well-characterized autoreactivity from antibodies encoded by the V(H)4-34 gene and for other hallmarks of an autoreactive repertoire are apparent mainly for class-switched B cells and not for IgM germinal center, IgM memory, or IgM plasma cells. Other possible indicators of autoreactivity found selected with immunoglobulin class include J(H)6 gene segment usage, increased frequency of B cells with long third hypervariable regions, and distal J(kappa) gene segment bias. Of particular interest is the finding that B cells with these same characteristics are selected into the lineage of B cells that have undergone the unusual class switch from constant region C mu to C delta (C delta-CS). The C delta-CS population also displays an increased frequency of charged amino acids localized to the complementarity-determining regions, further suggesting autoreactivity, and evidence is presented that these B cells had undergone extensive receptor editing. Thus, the C delta-CS lineage may be a "sink" for B cells harboring autoreactive specificities in normal humans. A model for a new tolerizing mechanism that could account for the C delta-CS lineage is presented.

Autonomous SHIP-dependent FcgammaR signaling in pre-B cells leads to inhibition of cell migration and induction of cell death.

Mature B cells express a single immunoglobulin Fc receptor, FcgammaRIIB, that functions to block downstream signaling by co-aggregated antigen receptors. Co-aggregation of receptors is essential because BCR activated kinases must phosphorylate FcgammaRIIB to recruit SHIP and mediate inhibitory signals. Pre-B cells also express FcgammaRIIB, but since they do not yet express antigen receptor, it is unclear when they are activated physiologically. Here, we demonstrate that aggregation of the FcR on pre-B cells leads to potent inhibitory signaling. Aggregation of the FcR alone leads to downstream effects including the induction of cell death and the blockade of SDF-1 induced migration. The biochemical circuitry that mediates this response is unique because although SHIP is required for this signaling and is phosphorylated upon receptor aggregation, this occurs in the absence of FcgammaRIIB phosphorylation. Results indicate that immune complexes may inhibit B cell production in the bone marrow by antigen non-specific mechanisms.

Cellular maturation defects in Bruton's tyrosine kinase-deficient immature B cells are amplified by premature B cell receptor expression and reduced by receptor editing.

In the mouse, Bruton's tyrosine kinase (Btk) is essential for efficient developmental progression of CD43(+)CD2(-) large cycling into CD43(-)CD2(+) small resting pre-B cells in the bone marrow and of IgM(high) transitional type 2 B cells into IgM(low) mature B cells in the spleen. In this study, we show that the impaired induction of cell surface changes in Btk-deficient pre-B cells was still noticeable in kappa(+) immature B cells, but was largely corrected in lambda(+) immature B cells. As lambda gene rearrangements are programmed to follow kappa rearrangements and lambda expression is associated with receptor editing, we hypothesized that the transit time through the pre-B cell compartment or receptor editing may affect the extent of the cellular maturation defects in Btk-deficient B cells. To address this issue, we used 3-83 mu delta transgenic mice, which prematurely express a complete B cell receptor and therefore manifest accelerated B cell development. In Btk-deficient 3-83 mu delta mice, the IgM(+) B cells in the bone marrow exhibited a very immature phenotype (pre-BCR(+)CD43(+)CD2(-)) and were arrested at the transitional type 1 B cell stage upon arrival in the spleen. However, these cellular maturation defects were largely restored when Btk-deficient 3-83 mu delta B cells were on a centrally deleting background and therefore targeted for receptor editing. Providing an extended time window for developing B cells by enforced expression of the antiapoptotic gene Bcl-2 did not alter the Btk dependence of their cellular maturation. We conclude that premature B cell receptor expression amplifies the cellular maturation defects in Btk-deficient B cells, while extensive receptor editing reduces these defects.

The porcine Ig delta gene: unique chimeric splicing of the first constant region domain in its heavy chain transcripts.

The pig delta gene is located approximately 3.4 kb downstream of the second transmembrane exon of the micro gene and shows a similar genomic structure to its counterpart in cow with three exons encoding the CH1, CH2, and CH3 domains. The porcine genomic deltaCH1 exon has been replaced by a recent duplication of the micro CH1 and its flanking sequences, a genetic event that also led to the formation of a short switch delta region, immediately upstream of the delta gene. The deltaCH1 exhibits a 98.7% similarity (314 of 318 bp) to the micro CH1 at the DNA level, whereas the homologies between the deltaCH2 and micro CH3, and the deltaCH3 and micro CH4 are only 33.3 and 35.8%, respectively. Either of the two CH1 exons ( micro and delta) could be observed in the expressed porcine IgD H chain cDNA sequences VDJ- micro CH1-H-deltaCH2-deltaCH3 or VDJ-deltaCH1-H-deltaCH2-deltaCH3, showing a pattern that has not been observed previously in vertebrates. In addition, transfection of a human B cell line, using artificial constructs resembling the porcine C micro -Cdelta locus, also generated both VDJ- micro CH1-deltaCH1-H1-deltaCH2 and VDJ -deltaCH1-H1-deltaCH2 transcripts. An examination of the pig delta genomic sequence shows a putative, second hinge region-encoding exon. Due to the lack of a normal branchpoint sequence for RNA splicing, this exon is not present in the normal pig delta cDNA. However, the exon could be spliced into most of the expressed transcripts in vitro in cell transfection experiments after introduction of a single T nucleotide to restore the branchpoint sequence upstream of the putative H2 exon.