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1.
Molecular cloning of a bovine immunoglobulin lambda chain cDNA 总被引:3,自引:0,他引:3
A cDNA library of the bovine mammary gland constructed in pBR322 was screened by mRNA hybrid-selected translation and by differential hybridization. Several immunoglobulin (Ig) lambda light-chain clones were identified and sequenced. Nucleotide sequence comparison of bovine and human Ig lambda chains showed a high degree of homology for constant regions and for J regions. The amino acid (aa) sequence encoded by the constant region of the bovine Ig lambda chain cDNA contains 107 aa with differences at 24 aa positions from the human Ig lambda chain. Three complementarity-determining regions (CDR1,2,3) characteristic of the variable region of bovine Ig lambda chain cDNA can be distinguished. The bovine and human sequences display good homology in the framework region 3 (FR3) but only patches of homology throughout the FR2 region. The 5' end of the bovine Ig lambda chain cDNA fragment of clone 1-14E contains five stop codons: two in CDR1, one in FR1 and two in the hydrophobic prepeptide region. These data suggest that the Ig lambda mRNA of clone 1-14E is transcribed from the V lambda pseudogene. 相似文献
2.
Cloning and sequencing of immunoglobulin variable-region genes using degenerate oligodeoxyribonucleotides and polymerase chain reaction 总被引:4,自引:0,他引:4
A procedure is described for using the polymerase chain reaction (PCR) to amplify and clone the cDNA from mouse immunoglobulin (Ig) variable (V) regions. This method uses a set of universal 5'-oligodeoxyribonucleotide primers that are degenerate and allow for the amplification of Ig V-region sequences from gamma and mu heavy chains and from kappa light chains. Selective first-strand cDNA synthesis is performed using Ig constant region primers and then a PCR is achieved by using the appropriate universal 5'-primer. The universal Ig heavy-chain primer was used to amplify the V-region cDNA from gamma and mu isotypes and the universal light-chain primer was used to amplify three separate kappa light V-region sequences. This procedure was used to obtain Ig V-region gene sequences from hybridomas secreting IgG1/kappa, IgG2b/kappa and IgM/kappa isotypes. 相似文献
3.
J S Andris P H Ehrlich L Ostberg J D Capra 《Journal of immunology (Baltimore, Md. : 1950)》1992,149(12):4053-4059
Structural studies of human antibody V regions have been largely limited to those involving the fetal repertoire, autoantibodies, and malignant cell rearrangements, leaving the "normal" repertoire relatively unexplored. In this study we describe the nucleotide sequences of the H and L chain V regions of four antibodies specific for the surface Ag of the hepatitis B virus. Monoclonal cell lines were derived from healthy individuals who received standard immunizations with the serum-derived or recombinant hepatitis B virus vaccines by fusion of PBL to a heterohybridoma cell line, SPAZ-4. We utilized the polymerase chain reaction to amplify the H and L chain V regions for cloning and sequencing. The four antibodies express the following V region combinations: VHIII/V lambda V, VHIII/V kappa II, VHIV/V kappa I, VHV/V lambda III. When compared to germline genes with the closest sequence homology, all of the V regions appear to have undergone somatic mutation, ranging from 3.4 to 11.3% for the H chain, and 5.1 to 9.2% for the L chain. Analysis of the mutations shows them to be typical for an Ag-driven immune response. 相似文献
4.
Analysis of 10 cDNA encoding lambda L chains of horse Ig indicated that this species may employ a relatively small number of variable region (V lambda) genes in the splenic B cell population. The V lambda sequences of all of the cDNA analyzed were closely related (> 88% identity at the nucleotide level) and were characterized by a deletion of the amino acid residue at position 3 compared with V lambda sequences so far described in other species. The 10 V lambda sequences could be grouped into three groups, V lambda 1 to V lambda 3, on the basis of a number of linked substitutions. Sequences within the groups showed the greatest divergence in the third cdr regions and at the V-J junctions. The junctional variation included amino acid substitutions on both sides of the V-J junction as well as the insertion or deletion of two to four amino acid residues. Four C lambda genes were identified in genomic blots of horse DNA, and three of these were found expressed in splenic cDNA. The fourth C lambda gene may represent a pseudogene, inasmuch as the associated J region possessed a defective heptamer joining sequence. Six of the nine possible V lambda-C lambda combinations were found in the cDNA analyzed, suggesting that genes belonging to groups V lambda 1 through V lambda 3 may rearrange to any one of three J lambda-C lambda genes. One V lambda germline gene was characterized and found to represent a distinct V lambda group (V lambda 4), not represented in the cDNA sequences analyzed. The number of germline V lambda genes was estimated to be 20 to 30, based on analysis of restriction fragments hybridizing with V lambda probes. On the basis of these data, we propose that the V lambda repertoire in horse may consist of relatively limited number of genes, of which only a few may be used at high frequency in the splenic B cell population. The results indicate that predominance of lambda-chains in horse Ig may not simply be due to the presence of a large germline V lambda gene repertoire. 相似文献
5.
Burkitt's lymphoma is a malignancy of mature B cells expressing somatically mutated V region genes. 总被引:4,自引:0,他引:4
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U. Klein G. Klein B. Ehlin-Henriksson K. Rajewsky R. K¼ppers 《Molecular medicine (Cambridge, Mass.)》1995,1(5):495-505
BACKGROUND: The developmental stage from which stems the malignant B cell population in Burkitt's lymphoma (BL) is unclear. An approach to answering this question is provided by the sequence analysis of rear-ranged immunoglobulin (Ig) variable region (V) genes from BL for evidence of somatic mutations, together with a phenotypic characterization. As somatic hypermutation of Ig V region genes occurs in germinal center B cells, somatically mutated Ig genes are found in germinal center B cells and their descendents. MATERIALS AND METHODS: Rearranged V kappa region genes from 10 kappa-expressing sporadic and endemic BL-derived cell lines (9 IgM and 1 IgG positive) and three kappa-expressing endemic BL biopsy specimens were amplified by polymerase chain reaction and sequenced. In addition, VH region gene sequences from these cell lines were determined. RESULTS: All BL cell lines and the three biopsy specimens carried somatically mutated V region genes. The average mutation frequency of rearranged V kappa genes from eight BL cell lines established from sporadic BL was 1.8%. A higher frequency (6%) was found in five endemic cases (three biopsy specimens and two BL cell lines). CONCLUSIONS: The detection of somatic mutations in the rearranged V region genes suggests that both sporadic and endemic BL represent a B-cell malignancy originating from germinal center B cells or their descendants. Interestingly, the mutation frequency detected in sporadic BL is in a range similar to that characteristic for IgM-expressing B cells in the human peripheral blood and for mu chain-expressing germinal center B cells, whereas the mutation frequency found in endemic BL is significantly higher. 相似文献
6.
A cDNA library was constructed from the mRNA of bovine mammary gland which contained Ig lambda producing plasmacytes. Overlapping clones encompassing the major portion of the coding sequence of the Ig lambda mRNA were isolated and sequenced. Predicted amino acid sequence shows a mature polypeptide of 217 residues in which V, J and C regions can be distinguished. The constant region has greatest homology with human Ig lambda mRNA constant region. The area of the V region between CDR3 and CDR2 has also great homology with the same area of human lambda chain. 相似文献
7.
Two isoforms of human membrane-bound alpha Ig resulting from alternative mRNA splicing in the membrane segment 总被引:2,自引:0,他引:2
L M Yu C Peng S M Starnes R S Liou T W Chang 《Journal of immunology (Baltimore, Md. : 1950)》1990,145(11):3932-3936
Antibodies specific for membrane-bound Ig (mIg) but not for their secreted forms would be useful not only for studying the function of mIg but also for modulating B cell activities in vivo. We have proposed that the extracellular portions of the membrane anchor peptides of mIg can be used as antigenic sites for isotype-specific targeting of B cells. Clones containing the genes of human Ig alpha 1 or alpha 2 subclasses were isolated from a genomic DNA library. The gene segments encoding the membrane peptides and their flanking regions were amplified by polymerase chain reaction, subcloned into plasmid pUC19, and the DNA sequences were determined. Human alpha 1 and alpha 2 genes, like murine alpha gene, each has only one membrane exon. The sequences of the human alpha 1 and alpha 2 genes are almost identical in the membrane peptide-coding region. The mRNA from a human mIgA-expressing B cell line, DAKIKI, was isolated, its cDNA prepared, and the segments spanning the membrane peptide-coding region and a part of the constant domain 3 amplified by polymerase chain reaction. DNA sequences revealed that there are two isoforms of alpha 1-chain, resulting from the alternative splicing of the third constant domain of H chain to two acceptor sites in the membrane exon. One isoform has a segment of 32 and the other 26 amino acid residues in the extracellular portion of the membrane peptide. These segments may serve as isotype-specific antigenic epitopes for antibody targeting of mIgA-bearing B cells. 相似文献
8.
P J Kelly V Pascual J D Capra P E Lipsky 《Journal of immunology (Baltimore, Md. : 1950)》1992,148(5):1294-1301
Polyclonal activation of human B cells is achieved by coculture with T cells stimulated by mAb to the CD3 molecular complex. By formal limiting dilution analysis, approximately 60% of human peripheral blood B cells were found to produce Ig in this system. When individual B cells were cultured in microtiter wells with anti-CD3-activated T cells, more than one-third of cultures producing Ig contained multiple Ig H chain isotypes. Similar results were observed when individual IgM-expressing B cells, selected and dispersed by FACS were cultured with anti-CD3-activated T cells. The clonality of the B cells producing multiple Ig isotypes was supported by L chain analysis of the secreted Ig. Of the wells containing more than one H chain isotype, nearly 85% contained only a single L chain type. Clonality was further examined by polymerase chain reaction amplification of cDNA harvested from cultures originally seeded with individual B cells. In general, only a single VH gene family could be amplified from cultures producing more than one Ig isotype. Three separate VH regions were cloned and sequenced. One, a VHIV-mu was nearly identical to a previously described VH gene VH71.4; as second, a VHIV-gamma was very similar to a previously described VH gene segment V-79, whereas a third, a VHIII-gamma differed by 14% in nucleotide sequence from its closest germline counterpart VH3005. These results indicate that anti-CD3-activated T cells not only stimulate the majority of B cells to secrete Ig, but also induce individual B cells to produce multiple Ig H chain isotypes. Additionally, the procedure described provides a reliable method to sample a large proportion of the human peripheral B cell repertoire. 相似文献
9.
Sequence of C region of L chains from Xenopus laevis Ig 总被引:4,自引:0,他引:4
D J Zezza C A Mikoryak J Schwager L A Steiner 《Journal of immunology (Baltimore, Md. : 1950)》1991,146(11):4041-4047
A cDNA expression library, prepared from Xenopus laevis splenocytes, was screened with antibodies to Xenopus Ig. One clone, lambda XIg23, reacted with antibodies to IgY and to IgM; the insert hybridized to approximately 1.3-kb RNA from spleen, the approximate size expected for L chain mRNA. An additional clone, lambda XIg31, was identified by cross-hybridization. The inserts of lambda XIg23 and lambda XIg31 begin in the third framework region of the V region and extend through the C region to the poly(A) tail. Except for a single nucleotide difference, the two C region sequences are identical. The amino acid sequence of the C region was compared with the sequences of a variety of C kappa and C lambda, as well as to C region sequences of L chains from Rana catesbeiana and from two species of shark. The Xenopus C region resembles mouse and human C kappa slightly more than C lambda. The similarity of the Xenopus and Rana C regions to each other is approximately the same as that of either amphibian sequence to mammalian CL. The data are discussed in terms of the evolution of kappa and lambda C regions. 相似文献
10.
Molecular cloning of a human immunoglobulin lambda chain variable sequence 总被引:10,自引:4,他引:6
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We have cloned a human V lambda cDNA sequence from an Ig lambda-producing human Burkitt lymphoma cell line (BL2) by taking advantage of a cloned constant region gene as a primer for cDNA synthesis instead of an oligo(dT) primer. The amino acid sequence deduced from the nucleotide sequence of V lambda clones is highly related to that of the NEW V lambda protein of subgroup I. Southern blot hybridization of human DNAs with the V lambda I probe showed at least 12 hybridizing V lambda fragments. These fragments are amplified in K562 cells which derive from a case of chronic myelogenous leukemia and contain an amplified c-abl oncogene and amplified C lambda sequences. 相似文献
11.
Isolation and sequence of sheep Ig H and L chain cDNA 总被引:3,自引:0,他引:3
Sheep lymphocyte poly(A+) RNA was used as a template for the enzymatic synthesis of cDNA before cloning into the expression vector lambda gt11. Screening of the cDNA library with mAb probes resulted in the isolation of two recombinant phages containing Ig coding sequences of 704 bp and 925 bp. These were inserted into the EcoRI site of pUC18 and named pSLC (sheep Ig L chain) and pSHC (sheep Ig H chain). The insert in pSLC revealed sequence homology by using GenBank to lambda L chain and pSHC revealed sequence homology to IgG sequences from various species. The L chain cDNA contained the full translation sequence and 5' and 3' nontranslating region while the H chain cDNA coded for the secreted form of IgG1 and lacked sequences upstream from the C region. The derived amino acid sequences showed significant homology with various Ig sequences already described for human, mouse, rabbit, pig, and chicken but the degree of homology showed no consistency with established phylogenetic relationships. 相似文献
12.
Somatic point mutations in unrearranged immunoglobulin gene segments encoding the variable region of lambda light chains. 总被引:3,自引:1,他引:2
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Somatic point mutations are usually found in the coding and flanking regions of functionally and aberrantly rearranged immunoglobulin variable region gene segments. Mutations in the unrearranged V gene segments of myelomas or hybridomas have not been described so far. We have cloned and sequenced unrearranged V lambda gene segments from several cell lines. There were no nucleotide changes in four unrearranged V lambda segments: one V lambda 1 from a lambda 3-producing hybridoma and one V lambda 2 from a lambda 1-producing myeloma (J558) and two V lambda 2 from a kappa-producing myeloma (P3X63). However, we found somatic mutations in the unrearranged V lambda segments from the lambda 2-producing myeloma MOPC315. The unrearranged V lambda 1 gene segment had two mutations in the coding region and the unrearranged V lambda 2 had one mutation in the 3' flanking region. We also cloned and sequenced the unrearranged J lambda and C lambda gene segments of MOPC315 and found no sequence alterations. This is consistent with the notion that the overall mutation rate is not higher in this cell line. Therefore, we suggest that the somatic hypermutation system can use unrearranged V gene segments as substrates. The extensive sequencing required for this work revealed a number of errors in the reported nucleotide sequences of the Ig lambda locus in BALB/c mice. 相似文献
13.
14.
Deletion of the immunoglobulin kappa chain intron enhancer abolishes kappa chain gene rearrangement in cis but not lambda chain gene rearrangement in trans. 总被引:16,自引:2,他引:14
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Immunoglobulins (Ig) secreted from a plasma cell contain either kappa or lambda light chains, but not both. This phenomenon is termed isotypic kappa-lambda exclusion. While kappa-producing cells have their lambda chain genes in germline configuration, in most lambda-producing cells the kappa chain genes are either non-productively rearranged or deleted. To investigate the molecular mechanism for isotypic kappa-lambda exclusion, in particular the role of the Ig kappa intron enhancer, we replaced this enhancer by a neomycin resistance (neoR) gene in embryonic stem (ES) cells. B cells heterozygous for the mutation undergo V kappa-J kappa recombination exclusively in the intact Ig kappa locus but not in the mutated Ig kappa locus. Homozygous mutant mice exhibited no rearrangements in their Ig kappa loci. However, splenic B cell numbers were only slightly reduced as compared with the wild-type, and all B cells expressed lambda chain bearing surface Ig. These findings demonstrate that rearrangement in the Ig kappa locus is not essential for lambda gene rearrangement. We also generated homozygous mutant mice in which the neoR gene was inserted at the 3' end of the Ig kappa intron enhancer. Unexpectedly, mere insertion of the neoR gene showed some suppressive effect on V kappa-J kappa recombination. However, the much more pronounced inhibition of V kappa-J kappa recombination by the replacement of the Ig kappa intron enhancer suggests that this enhancer is essential for V kappa-J kappa recombination. 相似文献
15.
E Paul A Livneh A J Manheimer-Lory B Diamond 《Journal of immunology (Baltimore, Md. : 1950)》1991,147(8):2771-2776
We report the cDNA sequence of an expressed human V lambda II gene and present an RFLP analysis of the Ig gene family defined by this clone. This V lambda II gene was expressed in a monoclonal B cell line generated from a patient with SLE by transformation with EBV. The encoded lambda L chain displays the 8.12 Id, an Id common to anti-DNA antibodies from patients with SLE. Using a coding region probe we estimate from Southern blot analysis that the germline V lambda II gene family contains at least 15 members. Many of the V lambda II restriction fragments are polymorphic both in SLE patients and in nonautoimmune individuals. EcoRI, HindIII, and TaqI RFLP analyses of the V lambda II gene family and EcoRI analysis of the C lambda gene family reveal no polymorphisms specific to SLE. Observed V lambda II and C lambda allele frequencies are the same among SLE patients and nonautoimmune individuals, and show no evidence of linkage disequilibrium between the two loci. 相似文献
16.
17.
Cloning and sequence analysis of an Ig lambda light chain mRNA expressed in the Burkitt''s lymphoma cell line EB4. 总被引:5,自引:1,他引:4
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A cDNA library was constructed from the mRNA of the Ig lambda producing Burkitt's lymphoma cell line, EB4. Overlapping clones encompassing the coding sequence of the Ig lambda mRNA were isolated and sequenced. The predicted amino acid sequence shows a short hydrophobic leader peptide and a mature polypeptide of 217 residues in which V, J and C regions can be distinguished. The V region belongs to subgroup VI and has greatest homology (80%) with the Amyloid-AR protein. The constant region is the Kern- Oz+ isotype. Probing normal human DNA with the subcloned V lambda coding sequence detects one gene at high stringency and a family of 11 members at low stringency. To date, no restriction enzyme site polymorphisms have been detected. The V lambda VI gene is rearranged on both chromosomes of EB4 and is deleted on both chromosomes in the Burkitt's lymphoma cell line BL2. 相似文献
18.
cDNA clones encoding rabbit immunoglobulin lambda chains. Evidence for length variation of the third hypervariable region and for a novel constant region.
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Five cDNA clones designated pDH2, pDH8, pDH9, pDH31 and pDH101 encoding rabbit immunoglobulin lambda light chain sequences have been characterized. Comparison of the V lambda sequences suggests that, in addition to an increased divergence in all of the complementarity-determining regions (CDRs), variable-region diversity is amplified by the length heterogeneity of the CDR3, at the V lambda-J lambda junction. An insertion of four codons at positions 48a-d has been noted in three cDNA sequences. This insert, not found in lambda nor kappa light chains of other species, has a variable sequence, suggesting its possible implication in expanding variability of the CDR2. One of the cDNA clones was shown to encode a novel C lambda region which differs by four amino acid substitutions from the C lambda region common to all the other clones. Thus, the rabbit can use two different C lambda genes, which might correlate with the expression of the two known allotypes of lambda chains, C7 and C21. Southern blotting experiments indicate a small number of germ-line V lambda genes and the cDNA nucleotide sequence data reported here suggest that several of these genes can be expressed. The possibility of at least two V-J-C gene clusters is discussed. 相似文献
19.
Somatic hypermutation of immunoglobulin kappa may depend on sequences 3'' of C kappa and occurs on passenger transgenes. 总被引:20,自引:4,他引:16
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We have compared the pattern of somatic mutation in different immunoglobulin kappa transgenes and suggest that an element(s) located between 1 kb and 9 kb 3' of C kappa is necessary for somatic hypermutation of the antibody V gene. The sequences of transgenic and endogenous Ig V regions were determined in antigen-specific B cell hybridomas specific for 2-phenyloxazolone from independent lines of hyperimmunized transgenic mice. We analysed somatic mutation of the transgene both in hybridomas in which the transgenic kappa chain contributes to the antigen combining site as well as in hybridomas in which the transgene is a passenger with the expressed antibody being composed of endogenously-encoded heavy and light chains. In both cases, nucleotide changes in the transgene are correctly targeted to the V region and are absent from the C region. They accumulate at a similar rate to that in the endogenous Ig genes within the same cell and we find that, irrespective of whether or not the transgene kappa is directly selected by antigen, somatic mutation occurs at a similar rate and involves only single base substitutions. Furthermore, the pattern of mutations in passenger transgenes gives information about the intrinsic sequence specificities of the somatic hypermutation mechanism. 相似文献