Structure and genomic organization of a second cluster of immunoglobulin heavy chain gene segments in the channel catfish

The structure, organization, and partial sequence of a 25-kb genomic region containing a second cluster of H chain gene segments in the channel catfish (Ictalurus punctatus) has been determined. Multiple VH gene segments, representing different VH families, are located upstream of a germline-joined VDJ. The VDJ segment has a split leader sequence and a single open reading consistent with that expressed in members of the VH1 family. Downstream of the germline-joined VDJ is a single JH segment and two pseudogene exons structurally similar to the Cmu1 and Cmu2 exons of the functional gene. Both pseudogene exons are multiply crippled with RNA splice sites destroyed, and open reading frames are interrupted by termination codons, insertions, and/or deletions. Sequence alignment of a 10.8-kb region within the second H chain cluster with the genomic sequence of the nine JH segments and the functional Cmu within the first H chain gene cluster indicates that the second H chain gene cluster probably arose by a massive duplication event. The JH region of the VDJ, the coding and flanking regions of the single JH segment, and the pseudogene Cmu exons were readily aligned with homologous segments in the first gene cluster. This duplication event may have extended to include the upstream VH segments. A member of the Tc1 mariner family of transposable elements is located downstream of the pseudogene Cmu2, which suggests that the transposition may have contributed to the evolution of the duplicated Cmu.     

Molecular characterization of five neutralizing anti-HIV type 1 antibodies: identification of nonconventional D segments in the human monoclonal antibodies 2G12 and 2F5

We have stabilized a panel of 33 hybridomas producing human monoclonal antibodies (MAbs) against HIV-1 gp160 and p24. Five of these antibodies were able to neutralize different HIV-1 isolates, and two of them (2F5 and 2G12) revealed remarkable potential to neutralize primary virus isolates of different clades in several in vitro tests. To determine whether a structural basis for neutralization could be identified, we analyzed the antibodies at the molecular level. This study reports the primary nucleotide and deduced amino acid sequences of the rearranged heavy and light chain V segments (VH, Vkappa) of the neutralizing MAbs (1B1, 1F7, 2F5, 2G12, and 3D5) and the nonneutralizing anti-gp41 MAb 3D6. Aligning the V segments with the nearest related germline genes illustrated the occurrence of somatic mutations. The neutralizing MAbs show mutational rates comparable to those of antibodies that appear in patients in whom the immune system is under constant antigenic pressure over a long period of time. In contrast, 3D6, which recognizes the immunodominant region on gp41, displays homologies as high as 97 and 98% compared with its VH and Vkappa germline genes. The diversity segments [D(H)] of 1B1, 1F7, 3D5, and 3D6 were assigned to single D(H) segments on the chromosomal D(H) locus. 2F5 presents a D(H) segment 52 nucleotides in length, which could be explained by fusion of two segments on the immunoglobulin heavy chain locus that have not yet been described as rearranged regions. 2G12 D(H) shows best homologies to a D(H) segment between D3-22 and D4-23. This D(H) segment could be the reason for the rare occurrence of antibodies competing with 2G12. Since this nearest related chromosomal region on the D(H) locus does not display recombination signals at the flanking regions, this segment is normally not taken into consideration as a site for immunoglobulin heavy chain rearrangement.     

Dobutamine stress echocardiogram: emergency department evaluation of chest pain

Secondary immune responses to T-independent antigens are characterized by little or no affinity maturation, a phenomenon attributed to limited somatic hypermutation. In the human immune response to Haemophilus influenzae type b capsular polysaccharide, however, there are numerous differences between rearranged heavy chain variable region gene segments and candidate germline genes, irrespective of antigen presentation in a T-independent or T-dependent form. To determine the characteristics of somatic hypermutation in this response, we analyzed rearranged heavy chain variable region segments and associated 3' untranslated JH4-JH5 introns from monoclonal anti-Hib PS antibodies. Mutation of untranslated introns and heavy chain variable segments in both T-independent and T-dependent responses resembles that described in murine and unselected human immune responses. Although mutation is frequent in both T-independent and T-dependent anti-Hib PS responses, there is little evidence of antigen-driven selection, suggesting that ongoing pressure to conserve the variable segment germline configuration limits affinity maturation in this immune response.     

Ig heavy chain of the sturgeon Acipenser baeri: cDNA sequence and diversity

To investigate the gene organization of the IGH locus, and the VH diversity of the Siberian sturgeon, a cDNA library was constructed and screened with VH-specific probes from two holostean fish. Isolated clones were analyzed and domain-specific probes used in rescreening of the library, Southern blot analysis, and northern blots. It was concluded that the Siberian sturgeon has one IGH locus with a translocon type of organization. Two allelic variants of the mu gene were found, with identities ranging from 80 to 100% for the different domains (highest for CH4 and lowest for CH2). Sturgeon CH sequences are most closely related to those of holostean fish. There are three distinct VH families, VHI grouping with mammalian clan III, VHII grouping with the teleost clan, and VHIII grouping with the archaic clan. The variability of the CDR 3 region is substantial, and we identified a number of conserved motifs in the D segment. Further, we deduced that there are at least nine different JH segments in the locus, contributing to the antibody repertoire of the sturgeon. The variable segments of the three families can be associated with any of the D or JH segments in the rearrangement. Sturgeon, in addition to the random rearrangement of VH, D, and JH segments, have exonuclease activity, and an introduction of N and probably P nucleotides at the site of rearrangement.     

The complete nucleotide sequence of the human immunoglobulin heavy chain variable region locus

The complete nucleotide sequence of the 957-kb DNA of the human immunoglobulin heavy chain variable (VH) region locus was determined and 43 novel VH segments were identified. The region contains 123 VH segments classifiable into seven different families, of which 79 are pseudogenes. Of the 44 VH segments with an open reading frame, 39 are expressed as heavy chain proteins and 1 as mRNA, while the remaining 4 are not found in immunoglobulin cDNAs. Combinatorial diversity of VH region was calculated to be approximately 6,000. Conservation of the promoter and recombination signal sequences was observed to be higher in functional VH segments than in pseudogenes. Phylogenetic analysis of 114 VH segments clearly showed clustering of the VH segments of each family. However, an independent branch in the tree contained a single VH, V4-44.1P, sharing similar levels of homology to human VH families and to those of other vertebrates. Comparison between different copies of homologous units that appear repeatedly across the locus clearly demonstrates that dynamic DNA reorganization of the locus took place at least eight times between 133 and 10 million years ago. One nonimmunoglobulin gene of unknown function was identified in the intergenic region.     

Use of D gene segments with irregular spacers in terminal deoxynucleotidyltransferase (TdT)+/+ and TdT-/- mice carrying a human Ig heavy chain transgenic minilocus

D gene segments with irregular spacers (DIR) are D gene segments that are specific to higher primates. Their use is controversial because of their G+C-rich long sequences. In the human, it has always been tempting to assume that a complementarity-determining region 3 sequence has been added by terminal deoxynucleotidyltransferase (TdT) activity and is not derived from DIR recombination. Herein, we examine the use of human DIR gene segments by cross-breeding the human Ig heavy chain minilocus pHC1 transgenic mice and TdT-deficient mice. In the absence of TdT and with a defined set of human D gene segments, it is relatively easy to demonstrate that DIR2 is used to form human Ig heavy chains, contributing to 7% of the human heavy chain rearrangements. VHDJH rearrangements (where H is heavy chain) in the minilocus TdT-/- mice use small portions of DIR2 located throughout the coding sequence. These results constitute the strongest evidence to date that DIR gene segments are used to form human antibodies. Additionally, we show that direct and inverted DIR2JH and VHDIR2 rearrangements occur in the minilocus transgenic mice. During these rearrangements, DM2 3' signal sequence and a new DIR2 5' signal sequence are used. These rearrangements generally follow the 12/23 recombination rule. Our results at the VHDJH, DJH, and VHD levels indicate that DIR2 is used to form human heavy chains in transgenic mice. The rearrangement of this gene segment likely involves, however, other mechanisms in addition to the classical VHDJH recombination.     

Sources of DNA for detecting B cell monoclonality using PCR

AIMS: To evaluate the polymerase chain reaction (PCR) demonstration of clonal immunoglobulin heavy chain gene rearrangements using routinely prepared, unstained, and stained formalin fixed, paraffin wax embedded tissue samples. METHODS: Extracts from (a) fresh frozen tissue samples, (b) unstained, and (c) haematoxylin and eosin stained formalin fixed, paraffin wax embedded 5 microns tissue sections from 42 cases of low grade B cell lymphoma, all shown to be monoclonal by Southern blot analysis, were analysed using PCR. Two regions of the variable segment of the immunoglobulin heavy chain gene were amplified (framework 2 to joining region [Fr2/JH] and framework 3 to joining region [Fr3/JH]). Twelve samples of reactive lymphoid tissue were studied as controls. Products from each case were directly compared on polyacrylamide gels. RESULTS: Using both primer combinations, monoclonality was detected in 38 of 42 (90%) cases using fresh material, 37 of 42 (88%) using unstained paraffin wax embedded samples, and in 35 of 42 (83%) cases using haematoxylin and eosin stained sections. No false positive results attributable to fixation, processing, or staining were identified, although the efficiency of amplification using the Fr2/JH primers was significantly reduced. CONCLUSIONS: PCR determination of B cell clonality using paraffin wax embedded material is sufficiently sensitive and reliable for use as a routine diagnostic adjunct to conventional morphological and immunocytochemical assessment of lymphoproliferative disease.     

Characterizing the target of acute stroke therapy

bcl-2/IgH fusion is considered a genetic error which occurs at the diversity (D) to joining (J(H)) stage of the gene rearrangement process in the immunoglobulin heavy chain (IgH) gene locus. Translocations of the bcl-2 protooncogene to the IgH locus at ontogenetically later IgH gene rearrangements are thought to represent exceptions. In the present study we analysed the junctional nucleotide sequence of 18 bcl-2/IgH fusion genes identifiable by polymerase chain reaction performed on DNA extracted from diagnostic lymph node tissue of 14 follicular lymphoma patients. In all clones studied, segments of variable length were found interposed between bcl-2 and J(H) gene sequences. Nucleotide sequence data analysis and comparisons performed with the corresponding germline sequences using the GenBank/EMBL database revealed the presence of D segments in most of the bcl-2/IgH fusion genes under study (13/18). By the same kind of computer-aided analysis, previously unrecognized D segments were identified in many published junctional sequences. These results suggest that bcl-2/IgH fusion events are very prevalent in rather more differentiated stages in B-cell ontogeny than previously recognized.     

Targeted insertion of a variable region gene into the immunoglobulin heavy chain locus

A mutant mouse strain has been generated in which a rearranged immunoglobulin heavy (H) chain variable (V) region gene is placed into the heavy chain locus in its natural position, replacing the JH elements. In homozygous mutant mice, essentially all B cells in the spleen express the transgenic VH region in their antibodies. The proper location of the transgene relative to the constant region genes allows it to participate in isotype switching and undergo somatic hypermutation. Immunoglobulin transgenic mice generated in this fashion by gene targeting should prove useful for the exploration of immunoregulatory mechanisms.     

Analysis of heavy and light chain pairings indicates that receptor editing shapes the human antibody repertoire

In the bone marrow, diversity in the primary antibody repertoire is created by the combinatorial rearrangement of different gene segments and by the association of different heavy and light chains. During the secondary response in the germinal centres, antibodies are diversified by somatic mutation and possibly by further rearrangements, or "receptor editing". Here, we have analysed the pairings of heavy and light chain variable domains (VH and VL) in 365 human IgG+ B cells from peripheral blood, and established that these pairings are largely random. The repertoire is dominated by a limited number of pairings of segments and folds. Among these pairings we identified two identical mutated heavy chains in combination with two different mutated light chains (one kappa and one lambda). This shows that receptor editing occurs in the human periphery and that the same antibody lineage can be subjected to both receptor editing and somatic hypermutation. This suggests that receptor editing may be used together with somatic mutation for the affinity maturation of antibodies. We also propose that receptor editing has shaped variable gene segment use and the evolution of V gene families.     

Polymorphism detection and sequence analysis of human T-cell receptor V alpha-chain-encoding gene segments

The T-cell receptor (Tcr) provides specificity for antigen recognition by its variable domain, primarily consisting of two germline encoded variable (V) region gene segments. Thus it has been suggested that inherited polymorphisms in the TCRV gene segments could contribute to differential immune responsiveness (e.g., autoimmunity) in human populations. In the present study, we have sought potentially functional polymorphisms in the germline TCRAV gene segments. Using denaturing gradient gel electrophoresis on polymerase chain reaction (PCR)-amplified products from the pooled DNA of many individuals, we identified polymorphisms in the TCRAV2S1, AV4S1, AV7S1, and AV8S1 gene segments. A complete DNA sequence analysis of these PCR products identified polymorphisms that affected amino acids in the predicted antigen-binding regions of the Tcr alpha chain, as well as polymorphisms in the introns. Genotype analysis of all nine DNA point mutations showed a 5%-50% range (averaging 35%) of minor allele frequencies, often resulting in individuals homozygous for the alternate allele forms. All possible haplotype combinations of the amino acid-affecting polymorphisms were found, indicating that in human populations there are a large number of different germline haplotypes encoding V gene segment alleles. These TCRAV coding region polymorphisms provide the rationale for, and allow the direct testing of, hypotheses concerning inherited polymorphisms within the T-cell receptor genes that may contribute to autoimmune susceptibility.     

Association of kinesin with microtubules in diverse cytoskeletal systems in the outer segments of rods and cones

The membranous outer segments of vertebrate photoreceptors are supported by cytoskeletons consisting of microtubules and associated proteins, which occur as the ciliary axoneme in rods and cones, and as a separate cytoskeletal system at the incisures of rod outer segments. We performed an immunocytochemical study of the cytoskeleton in photoreceptors isolated from amphibian retinas and found that immunoreactivity to the heavy chain of the motor protein kinesin was closely associated with the microtubules in each of these outer segment cytoskeletal systems. In the outer segments of cones, kinesin heavy chain immunoreactivity was confined to a streak at the axoneme that extended to the outer segment tip. In the outer segments of rods, kinesin heavy chain immunoreactivity was found as both a short streak at the axoneme and a series of long parallel lines that coincided with the microtubules at rod outer segment incisures. Our findings constitute the first report of kinesin in the axoneme of cones and at the incisures of rods. Closely associated with microtubules, kinesin in photoreceptor outer segment axonemes and at rod outer segment incisures can transport materials longitudinally along the microtubules and/or connect these with each other and/or with other components. Because these cytoskeletal systems differ in fundamental ways, kinesin can play different roles in each case, e.g., kinesin at rod outer segment incisures can have structural and functional roles that are unique to rods. These findings may have clinical relevance because similar cytoskeletal systems are expected to occur in the outer segments of human photoreceptors; thus, a disturbance involving kinesin in the cytoskeletal systems at photoreceptor axonemes and/or at rod outer segment incisures could interfere with the normal structure and function of photoreceptors and contribute to human photoreceptor degenerations.     

Evidence for immunoglobulin heavy chain variable region gene replacement in a patient with B cell chronic lymphocytic leukemia

Immunoglobulin heavy chain variable (V) gene replacement is an unusual recombinatorial event characterized by rearrangement of a germline V gene to a preformed VDJ gene complex. This phenomenon has occasionally been implicated in the emergence of clonal subpopulations during the course of acute lymphoblastic leukemia; it has also been found in murine precursor B cell lines. V gene replacement has never been described in lymphoproliferative disorders corresponding to more differentiated stages of B cell ontogeny. The present communication provides evidence for the operation of the same mechanism in B cell chronic lymphocytic leukemia (B-CLL). Genomic DNA and total cellular RNA extracted from peripheral blood mononuclear cells of a 48-year-old female patient diagnosed as having typical B-CLL were subjected to polymerase chain reaction (PCR) amplification aiming to detect rearranged clonal heavy and light chain variable genes (VH and VL, respectively). PCR consistently gave two VH amplification products, both at the DNA and the RNA level; similar analysis for the VL region revealed the presence of a single rearranged VK gene. Direct sequence analysis of the PCR products revealed that, except for a number of silent mutations, the single rearranged VK gene was identical to the germline A1-A17 VK gene. The two rearranged VH gene segments belong to the VHl and VHIII gene families and are closely homologous, respectively, to the germline gene segments V1-18 and V3-30, which have been shown to be used by autoantibodies. Both rearranged VH genes showed identical in-frame D-N-JH junctions and JH gene usage (JH5b), whereas the VH-N-D junctions were different. The above findings indicate that, during the course of the disease of our patient, VH gene replacement took place giving rise to two different clonally related subpopulations. This raises the intriguing possibility that the recombinase machinery, which governs Ig recombinatorial processes, might be operative even at more advanced stages in B cell ontogeny.     

Vectors for the expression of PCR-amplified immunoglobulin variable domains with human constant regions

Cassette vectors have been constructed for mammalian expression of complete immunoglobulin heavy and light chain genes whose variable regions are produced by the polymerase chain reaction (PCR). The light and heavy chain vectors have promoter, leader, partial intron, enhancer and constant region segments within modified pSV2-gpt and pSV2-neo plasmids, respectively. Variable (V) regions are obtained by PCR using a two step process: 1) the V gene is amplified from genomic or cDNA, cloned into an intermediate vector and sequenced; 2) the first PCR product serves as the template for a second amplification in which restriction enzyme recognition sites and limited flanking intron sequence are added. The second PCR product is inserted into the expression vector, which is then transfected into mouse myeloma cells. These vectors contain human constant regions and may be used to express chimeric, humanized or human Ig genes. This report describes the design of these vectors and their application for the expression of chimeric 60.3, an anti-CD18 antibody.     

A single predominantly expressed polymorphic immunoglobulin VH gene family, related to mammalian group, I, clan, II, is identified in cattle

In order to understand the generation of antibody diversity in cattle, seven cDNAs, from heterohybridomas secreting bovine IgM and IgG1 antibodies, were cloned and structurally analyzed for rearranged bovine VDJ genes. All of the seven bovine VH genes, together with four available bovine VH gene sequences, shared a high nucleotide sequence homology (84.2-93.5%). Based upon the criteria of nucleic acid homology > or =80%, all of the bovine VH gene sequences isolated from the expressed antibody repertoire constitute a single VH gene family, which we have designated as bovine VH1 (Bov VH1). An analysis of 44 bovine IgM-secreting mouse x cattle heterohybridomas, originating from polyclonally-activated PBLs from bovine leukemia virus-infected cattle, revealed that all of these expressed Bov VH1 (100%) based upon DNA sequencing and Northern dot blot. The bovine VH genes showed highest DNA sequence similarity, ranging between 81.5 and 87.6%, with a single sheep VH gene family (related to human VH4) and are, thus, closest to the VH genes from another ruminant species. The Bov VH1 gene family is most homologous to the murine VH Q-52 (71.8-78%) and human VH4 (67.4-69.8%) gene families, which belong to mammalian group, I, clan, II, VH genes. The CDR3 length of rearranged bovine VDJ genes is characteristically long (15-23 amino acids). The bovine JH gene segments were most homologous to human JH4 (82.1-87.2%) and JH5 (84.6-89.7%) genes, suggesting the existence of at least two JH gene segments. An analysis of CDRs provides evidence that somatic hypermutations contribute significantly to the generation of antibody diversity in cattle. Southern blot analysis of BamH I, EcoR I and Hind III digested genomic DNA from four cattle breeds (Holstein, Jersey, Hereford and Charolais) revealed three RFLP patterns; the genomic complexity of Bov VH1 ranged between 13 and 15 genes. These observations provide evidence for polymorphism at the bovine Ig-VH locus, similar to that seen in mice and humans.