Cloned MPC 11 myeloma cells express two kappa genes: a gene for a complete light chain and a gene for a constant region polypeptide.

Cloned MPC 11 mouse plasmacytoma cells synthesize a complete kappa light chain and also a kappa light chain constant region fragment. Partial amino terminal sequences of the in vitro forms of these two proteins have been determined. Both in vitro products contain typical light chain leaders; leaders are defined as the amino terminal sequences present on in vitro products but absent from the in vivo products found in living cells. The in vitro form of the MPC 11 complete light chain contains a leader sequence plus variable and constant region sequences. The in vitro form of the MPC 11 light chain constant region fragment contains a different leader sequence attached directly to a complete constant ragion sequence and has no variable region sequences. Thus the MPC 11 light chain fragment is not a degradation product of the MPC 11 complete light chain (or of any other complete light chain) and must be coded by a separate gene. The results reveal two unusual features of MPC 11 cells: first, expression of a unique variant light chain gene coding the light chain constant region fragment, and second, expression of two different kappa light chain genes (coding the complete light chain and the variant constant region fragment) in a single cell. In addition, evidence is provided that the in vitro forms of kappa light chains, three of which are presented here for the first time, include a minimum of three partially homologous but quite different leader sequences.     

Cloning and sequence of the cDNA corresponding to the variable region of immunoglobulin heavy chain MPC11.

Poly(A)-containing mRNA from mouse myeloma MPC11 was transcribed into cDNA which was cloned in the PstI site of the plasmid pBR322. The transformants were screened by hybridization with a cDNA fragment, derived from plasmid p gamma(11)7, corresponding to the 5' portion of the constant region of MPC11 heavy chain. Several positive transformants were found to contain various lengths of the variable region of the heavy chain. We describe the structure and sequence of one of these clones, pV(11)2, which contains cDNA corresponding to the entire variable region of MPC11 heavy chain and extends to codon 248 in the constant region. The protein sequence deduced from the DNA sequence indicates that the variable region of MPC11 heavy chain contains 121 amino acids and belongs to subgroup II of mouse heavy chains. Comparison of this sequence with other heavy chain sequences suggests a J (joining) segment of 16 residues which overlaps five residues of the third hypervariable region. The cDNA sequence shows that there is no discontinuity between the end of the variable region and the beginning of the constant region.     

Identification of a novel substitution in the constant region of a gene coding for an amyloidogenic kappa1 light chain.

Current concepts regarding the association between immunoglobulin (Ig) light chain structure and AL amyloidosis (AL) emphasize Ig variable region amino acid substitutions because the majority of light chain amyloid fibrils that have been sequenced contain amino termini of the variable region with only small amounts of the constant region. In this report, we describe a patient with rapidly progressive AL whose amyloid deposits contained primarily monoclonal kappa light chain constant region fragments. We sequenced and analyzed this AL protein, determining that it was an O18-O8 kappa1 variant and that the constant region possessed an unusual Ser-->Asn substitution at position 177. Using pre-mortem bone marrow cells, we cloned and sequenced the cDNA for this AL protein (HCAK1) and, using DNA from post-mortem somatic tissue, we cloned and sequenced the patient's kappa germline O18-O8 donor and kappa constant region (Ckappa) gene segments. The cDNA that coded for HCAK1 contained a variable region that was derived from O18-O8, showing 96.1% homology to germline, and a Ckappa that had a nucleotide substitution (AGC to AAC), resulting in the 177Ser-->Asn replacement. Two Ckappa genes were cloned from somatic tissue DNA, one identical to a known Ckappa sequence and another containing this substitution which likely is a new Ckappa allotype. Our findings indicate that further investigation is warranted into the contributions genetic polymorphisms and light chain constant regions may make to amyloidogenesis.     

DNA sequence of the constant gene region of the mouse immunoglobulin kappa chain.

The constant (C; ref. 3) gene segment of the immunoglobulin kappa light chain and about 1 kb upstream as well as downstream of the segment have been sequenced. The sequences of the C gene segment itself and parts of the upstream region were determined both in liver and in myeloma T DNA clones derived from the same mouse inbred strain. The sequences were identical, i.e. no somatic mutations were detected. Two sites in the region not coding for protein are discussed as possible targets of aberrant variable (V) gene translocations. Doublet frequencies were calculated in the approx. 2500 bp of the C region sequence reported in this paper and in the approx. 3400 bp of two rearranged V gene regions.     

Cell-type preference of immunoglobulin kappa and lambda gene promoters.

Immunoglobulin gene constant regions are known to be associated with strictly tissue-specific enhancer elements. Until recently the promoter of the variable region, which becomes linked to the constant region by somatic rearrangement, could have been viewed as a passive recipient of the enhancer stimulus. Here we show that the promoters of the immunoglobulin kappa and lambda light chain genes are approximately 20-30 times more active in lymphoid cells than in non-lymphoid cells. To avoid the problem of differential mRNA stability upon transfection of immunoglobulin genes into non-lymphoid cells we have constructed chimeric genes. All kappa mRNA sequences were progressively deleted to fuse the kappa gene promoter to a globin gene coding body. A similar chimeric gene was constructed with the promoter of the lambda gene. The cell-type preference of the promoter may be exploited during B-lymphocyte differentiation to regulate the immunoglobulin gene promoter independently from the enhancer.     

Isolation and analysis of recombinant DNA molecules containing yeast DNA.

2500 recombinant plasmids containing insertions of yeast nuclear DNA have been cloned in Escherichia coli. It can be calculated that about 85% of the yeast genome is represented in this collection. The clones have been characterized by hybridization to purified RNA species. Of the 2000 clones examined, 75 contain insertions of yeast ribosomal DNA, 201 contain insertions of yeast tRNA genes, and 26 contain DNA sequences that are complementary to abundant mRNA species.     

Molecular characterization of a major autoantibody-associated cross-reactive idiotype in Sjogren's syndrome

Primary Sjogren's syndrome is an autoimmune disorder characterized by lymphocytic infiltration of the salivary and lacrimal glands, producing associated dry eyes (keratoconjunctivitis sicca), dry mouth, and intermittently swollen salivary glands. A high proportion of the infiltrating B lymphocytes express surface and cytoplasmic Ig bearing a kappa-L chain-associated CRI defined by reactivity with the murine mAb, 17.109. To determine the structural basis for CRI expression in this disease, we generated CRI+ lymphoblastoid cell lines and a cDNA library from lymphocytes extracted from Sjogren's syndrome patients' salivary gland biopsy specimens. Nucleic acid sequence analyses of the mRNA of one such 17.109-CRI+ lymphoblastoid cell line (NOV) reveals the expressed kappa light chain variable region gene (V kappa gene) to be homologous to Humkv325, a conserved V kappa gene used at relatively high frequency in certain B cell malignancies. In addition, synthetic oligonucleotides, corresponding to the first and third frameworks and the second complementarity determining region of the Humkv325 gene, were used to identify and isolate clones from a cDNA library generated from SS salivary gland lymphocytes. Clones annealing specifically with one or more of these oligonucleotide probes contained kappa light chain cDNA. The sequences corresponding to the variable region of two clones (Taykv320 and Taykv306) were homologous to Humkv325. The V kappa genes of four other cDNA clones (Taykv322, Taykv310, Taykv308, and Taykv312) most likely were generated somatically from the rearranged Humkv325 gene through a limited number of nucleic acid base substitutions. Our results suggest that the high frequency of 17.109-CRI expression in Sjogren's syndrome patients results from a multiclonal expansion of B cells using Humkv325, and that the expressed Humkv325 may undergo somatic diversification in an apparent Ag-driven response.     

Partial amino acid sequence of a rabbit immunoglobulin light chain of allotype b5

The amino acid sequence of a rabbit immunoglobulin light chain of allotype b5 has been nearly completed. A comparison of its structure with that of light chains of allotypes b4, b6, and b9 confirms that the constant regions of these various kappa chains differ by 20-35%. The substitutions are clustered in parts of the second half of the chain, and the b5 form bears more resemblance to the b6 chain than to any other, in good agreement with previous serological data. The analysis of the variable region reveals the existence of certain allotype-associated residues which have also been reported in other b5 chains, but not in proteins of the other allotypes. An examination of the rabbit light chain sequences between positions 96 and 107 suggests that this portion of the chain may be encoded separately by a joining "J" DNA segment, as has been described previously for murine and human immunoglobulins. In the rabbit, however, these J kappa regions appear to differ from one allotype to another. Together with the extensive variations of the constant regions, these data suggest that the rabbit kappa gene organization more closely resembles the murine gamma system (four different C gamma genes each flanked by its J segment) than the murine kappa system (a single C kappa gene).     

Two V kappa germ-line genes related to the GAT idiotypic network (Ab1 and Ab3/Ab1') account for the major subfamilies of the mouse V kappa-1 variability subgroup

V kappa Ig germ-line genes have been isolated from recombinant clones prepared in separate libraries constructed from adult BALB/c liver DNA. Three different clones that strongly hybridized with a V kappa-GAT-specific probe were completely characterized and sequenced. All three genes exhibited common characteristic features in their sequences encompassing the 5' to the 3' noncoding region, with coding sections 95% homologous. A comparison with other V kappa genes shows that the size of the first intron is variability subgroup specific. Moreover, a direct correlation exists between the size of this intron and the entire length of the coding region. Nucleotide sequences of these genes were compared with V kappa chains expressed at the Ab1 and Ab1' levels of the GAT idiotypic network: Ag----Ab1----Ab2----Ab3 (Ab1'). K1A5 and K5.1 genes account for V kappa chains in Ab1 and Ab1' hybridomas, respectively. The high conservation of Ab1' sequences in light chain was also recently reported for the heavy chains, suggesting that immunization with Ab2 (anti-idiotypic) antibodies preferentially stimulates the direct expression of germ-line genes. K5.1 and K1A5 genes belong to the V kappa-1 variability subgroup and encode, without any amino acid substitution, V kappa domain in myeloma TEPC 105 and MOPC 467, which are V kappa-1A and V kappa-1C subgroup prototypes, respectively. These genes are extensively used in different mouse strains and in a number of antibodies of discrete specificities, such as anti-GAT, anti-DNP, anti-flagellin, anti-phosphorylcholine, anti-digoxin, anti-phenyloxazolone, and anti-DNA.     

Isolation of messenger RNA for an immunoglobulin kappa chain and enumeration of the genes for the constatn region of kappa chain in the mouse

The messenger RNA for an immunoglobulin light (kappa) chain was isolated from the mouse myeloma MOPC41 and shown to be almost twofold longer than necessary to code for its protein product. DNA complementary to the mRNA was synthesized with the RNA-directed DNA polymerase of Rous sarcoma virus. Although the individual chains of the cDNA² contained an average of only 270 nucleotides, the cDNA was heterogeneous in molecular weight, allowing the isolation of a fraction of the cDNA 620 nucleotides long. This large cDNA would be long enough to code for nearly all (95%) of the constant region if all the untranslated region of the mRNA were between the 3′ terminal poly(A) and the constant region. On the other hand, if all the untranslated region of the mRNA were at the 5′ terminus, this cDNA would code for 93% of the entire kappa chain.The specificity of nucleotide sequences in the cDNA was documented by molecular hybridization with both template RNA and RNA from various myelomas. The amount of hybridization obtained with myeloma RNA was approximately proportional to the amount of kappa chain protein produced by the various myeloma cells. In addition, there was no hybridization with RNA isolated from either BALB/c mouse liver or Escherichia coli.The genes for the constant region of the kappa chain were enumerated in the mouse genome by annealing cDNA to DNA from mouse liver and MOPC41 myeloma. The haploid genome of both tissues contained three to four genes for the constant region of kappa chain even when tested under conditions that would detect distantly related nucleotide sequences. The fact that there are only a few genes coding for the constant region of kappa chains implies that specialized genetic mechanisms are required for the generation of antibody diversity.     

Synthesis of a carboxyl-terminal (constant region) fragment of the immunoglobulin light chain by a mouse myeloma cell line

The MPC11 mouse myeloma cell line synthesizes not only heavy chains and light chains but also an 11,600 molecular weight light chain fragment. The fragment comprises 1% of the newly synthesized protein, compared to 8% for the complete light chain. Similar amounts of fragment are produced by a number of heavy plus light chain producing subclones, 18 independently generated light chain producing variant clones, and five independent non-producing variant clones. For both the heavy plus light chain producing and the non-producing cell types, less than 20% of the fragment appears to be secreted, while the remainder is metabolized with a half-life of 30 minutes. Radiochemical peptide analyses and radiochemical amino -terminal sequence analyses are consistent with the fragment containing most of the peptide sequences present in the carboxyl-terminal half (constant region) of the parent kappa light chain, but none of the variable region peptides. The fragments produced by a heavy plus light chain producing clone and a non-producing variant clone were identical by radiochemical peptide analysis. The results suggest that the constant region fragment may be a primary gene product, and in addition, they raise the possibility that the fragment may be specified by a gene discrete from the gene specifying a light chain.     

Untranslated immunoglobulin kappa light chain mRNA in a lambda light chain-producing mouse myeloma, MOPC104E

Fourteen clones were isolated in culture from a mouse myeloma, MOPC104E. All clones had kappa and lambda types of light chain mRNAs in approximately equimolar quantity as assayed by hybridization with specific complementary DNA (cDNA). However, the myeloma produces and secretes only lambda-type light chain protein. Both kappa- and lambda-type mRNAs in these clones were indistinguishable from kappa- and lambda-type mRNAs of other myelomas with respect to (a) adsorption to oligo-(dT) cellulose, (b) molecular size (12.6 S), and (c) thermal stability of the hybrids formed with corresponding cDNA. The kappa chain mRNA of MOPC104E cells, however, was translated very inefficiently both in vivo and in vitro, whereas the lambda chain mRNA was translated efficiently. These results indicate that each cell of MOPC104E myeloma synthesizes a crippled kappa chain mRNA in addition to a normal lambda chain mRNA.     

Cloning of V region fragments from mouse liver DNA and localization of repetitive DNA sequences in the vicinity of immunoglobulin gene segments.

Two different kappa light chain genes have previously been isolated from one mouse myeloma. The V (variable, abbreviations in ref. 2) gene segments of the two genes were now used to identify their germline counterparts in EcoRI digests of mouse liver DNA. In addition two sets of related V gene segments were found which hybridize with either of the two DNA probes. Five of the V region fragments of one set were cloned in a lambda phage vector and partially characterized by restriction mapping and Southern blot hybridization. Repetitive DNA sequences were found on each of the five fragments as well as on other cloned immunoglobulin gene containing fragments. Cross-hybridization between some but not all of the regions containing repetitive DNA sequences was observed.     

cDNA clones encoding rabbit immunoglobulin lambda chains. Evidence for length variation of the third hypervariable region and for a novel constant region.

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.     

鼻咽癌恶性转化基因Tx核苷酸序列分析

从人类鼻咽癌细胞系CNE2基因组DNA文库中克隆一个恶性转化基因Tx转染小鼠JB6 + 细胞 ,使细胞恶性转化 ,宿主细胞具有停泊非依赖生长的特性 .已经报道的Tx中一个 3 0kb片段(Tx3 0 )的序列分析结果表明 ,Tx包含人类免疫球蛋白κ轻链完整的J区 .进一步对Tx全长进行亚克隆和测序 ,并采用生物信息学方法进行分析表明 ,Tx包含人类Igκ完整的J区、C区基因片段、还有 5个重组信号序列 ,1个核基质结合序列和 1个N segment的插入 .Tx与正常人IgκJ、C区比较只是在某些位点存在碱基的缺失、插入或置换 ,二者同源性高达 98% ,其电子定位于 2p11 2 ,这与人Igκ的染色体定位是一致的 .可以认为 ,Tx是一个缺乏V区的异常的人类免疫球蛋白kappa轻链基因     

Human lupus anti-DNA autoantibodies undergo essentially primary V kappa gene rearrangements.

We have recently characterized the heavy chain variable region (VH) genes expressed by a panel of human anti-DNA antibodies derived from four patients with systemic lupus erythematosus and expressing an idiotypic marker representative of a subset of pathogenic autoantibodies. Here, we have cloned and sequenced the kappa chain variable region genes (V kappa) of the clones whose VH genes had been previously analysed. All the V kappa genes utilized map to the 280 kb portion of the 3' end of the locus, suggesting that they represent essentially the products of primary rearrangements. This proximal clustering of the V kappa genes used contrasts with the broad distribution of immunization-induced human antibody V kappa genes over 1400 kb of the locus. In addition, lupus autoantibodies show no tendency to express the downstream junctional (J kappa) exons--another indication of infrequent secondary variable gene assembly. Since successive rearrangements may extinguish high-affinity recognition of self antigens, we propose that this bias in V kappa and J kappa expression reflects a low rate of secondary light chain rearrangements among lupus autoantibodies. We also postulate that the corrective mechanism capable of editing potentially aggressive, self-reactive antibodies in these patients may be deficient--a deficit that could be genetically determined and/or somatically acquired.     

Cloning,sequence and variability analysis of expressed immunoglobulin light chain genes from yellowtail Seriola quinqueradiata

The cDNA clones encoding immunoglobulin (Ig) light (L) chain variable (V) region associated with constant (C) region were isolated from yellowtail (Seriola quinqueradiata) kidney by expressed sequence tag analysis (accession numbers: AB062619-AB062668, AB064322). The sequences of both VL and CL region contain well-conserved cysteine residues important for intra- and inter-domain interaction in mammals. Comparisons of the amino acid sequence of the CL domain with those of other species showed a high degree of similarity, with 88.3%, 59.8%, and 60.6% to those of wolf fish (Anarhichas lupus), rainbow trout IgL I isotype (Oncorhynchus mykiss) and channel catfish G isotype (Ictalurus punctatus), respectively. Multiple sequence alignments of the CL domain with those of higher vertebrates, however, did not readily allow it to be classified as kappa or lambda isotypes. Furthermore, the pI, hydrophobicity and variability of yellowtail VL regions were studied in 65 cDNA clones and the diversity was observed in CDR1, CDR2 and CDR3 regions.     

Cloning of immunoglobulin kappa light chain genes from mouse liver and myeloma MOPC 173

The organization of the kappa chain constant region gene was compared in DNA from an immunoglobulin-producing mouse myeloma (MOPC 173) and from liver. In situ hybridization using the Southern blotting technique revealed constant region gene-containing EcoRI-DNA fragments of 14 and 20 kb in the myeloma tissue whereas one EcoRI-DNA fragment with a length of 15 kb was found in liver DNA. After enrichment by RPC-5 chromatography and preparative electrophoresis the 14 kb fragment from MOPC 173 DNA and the 15 kb fragment from liver DNA were cloned in the bacteriophage lambda vector Charon 4A using in vitro packaging. Extensive characterization of the two fragments by restriction endonuclease mapping, in situ hybridization, and electron microscopy (R-loop and heteroduplex) showed that both fragments contain the constant region but no MOPC 173 variable region gene. Both fragments are homologous over a length of 12.5 kb including the constant region but differ from one another starting about 2.7 kb from the 5' end of the constant region gene. This indicates that the 14 kb EcoRI-DNA fragment from the myeloma tissue clearly resulted from somatic DNA rearrangement although it does not seem to carry the MOPC 173 variable region gene. These observations suggest that somatic DNA rearrangement of immunoglobulin light chain genes can involve both homologous chromosomes.Images     

Mutations, duplication, and deletion of recombined switch regions suggest a role for DNA replication in the immunoglobulin heavy-chain switch.

The heavy-chain switch from immunoglobulin M (IgM) expression to IgA expression is mediated by a recombination event between segments of DNA called switch regions. The switch regions lie two to six kilobases upstream of the mu and alpha constant region coding segments. Switch recombination to IgA expression results in a recombinant mu-alpha switch region upstream of the expressed alpha constant region gene. We have characterized the products of switch recombination by a lymphoma cell line, I.29. Two sets of molecular clones represent the expected products of simple mu to alpha switches. Five members of a third set of molecular clones share the same recombination site in both the mu and the alpha switch regions, implying that the five molecular clones were derived from a single switch recombination event. Surprisingly, the five clones fall into two sets of sequences, which differ from each other by several point mutations and small deletions. Duplication of switch region sequences are also found in these five molecular clones. An explanation for these data is that switch recombination involves DNA synthesis, which results in nucleotide substitutions, small deletions, and duplications.