Binary specification of nonsense codons by splicing and cytoplasmic translation.

Premature translation termination codons resulting from nonsense or frameshift mutations are common causes of genetic disorders. Complications arising from the synthesis of C-terminally truncated polypeptides can be avoided by 'nonsense-mediated decay' of the mutant mRNAs. Premature termination codons in the beta-globin mRNA cause the common recessive form of beta-thalassemia when the affected mRNA is degraded, but the more severe dominant form when the mRNA escapes nonsense-mediated decay. We demonstrate that cells distinguish a premature termination codon within the beta-globin mRNA from the physiological translation termination codon by a two-step specification mechanism. According to the binary specification model proposed here, the positions of splice junctions are first tagged during splicing in the nucleus, defining a stop codon operationally as a premature termination codon by the presence of a 3' splicing tag. In the second step, cytoplasmic translation is required to validate the 3' splicing tag for decay of the mRNA. This model explains nonsense-mediated decay on the basis of conventional molecular mechanisms and allows us to propose a common principle for nonsense-mediated decay from yeast to man.     

Human immunoglobulin kappa light chain genes of subgroups II and III.

The first complete sequences of functionally rearranged VK genes (abbreviations ref. 1) of subgroups II and III are reported. The genes have been cloned from lymphoid cell lines synthesizing KII or KIII light chains as evidenced from immunochemical analyses with anti-VK subgroup-specific antisera. These data, together with the sequence of a KIV gene (described in the accompanying paper) and those of previously published KI genes make possible a comparison of genes representative of the four known V region subgroups of human K light chains. The VKII gene is distinguished from the VKI, VKIII, and VKIV genes by a much longer intron within the leader sequence: 426 bp vs ca. 120-220 bp. Blot hybridization experiments with human DNA digests using probes from the KII and KIII genes and from the respective upstream regions help to define subgroup specific probes and hybridization conditions.     

Two rearranged immunoglobulin kappa light chain genes in one mouse myeloma.

The organization of immunoglobulin gene segments coding for kappa light chains has been studied in uncloned and cloned DNA from mouse liver and a mouse myeloma. It is known that the C (constant, ref. 2) gene segment is present in the tumor DNA on two EcoRI fragments of 14 and 20 kb and in liver DNA on a 15 kb fragment. The 14 kb myeloma and the 15 kb liver fragment have been cloned previously. Here we report on the cloning of the 20 kb myeloma fragment and present detailed restriction maps covering about 22 kb of DNA surrounding the C gene segment in liver and tumor DNA. The region on the 20 kb fragment has been localized where a DNA rearrangement had occurred. The presence of two rearranged kappa light chain genes in one tumor is discussed in regard to the molecular basis of allelic exclusion.     

Evolutionary relationship between human and mouse immunoglobulin kappa light chain variable region genes

Similar to the Igh-V multigene family, the human or mouse Igk-V repertoirer is a distorted continuum of homologous genes that may be grouped into families displaying >80% nucleic acid sequence similarity among their members. systematic interspecies sequence comparisons reveal that most human Igk-V gene families exhibit clear homology to mouse Ogk-V families (sequence similarity >74%). A hypothetical phylogenetic tree of Igk-V genes predicts that a minimum of seven Igk-V genes/families predate mammalian radiation. In two cases, several interrelated mouse Igk-V families exhibit phylogenetic equidistance with just one human Igk-V family, implying a more pronounced divergence for the elevated number of Igk-V gene families in the mouse. Mouse-human Igk-V comaprisons, moreover, illustrate how expansion, contraction, and perhaps deletion of Igk-V gene families shape the Igk-V repertoire during mammalian evolution.     

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     

B cell development in mice that lack one or both immunoglobulin kappa light chain genes.

We have generated mice that lack the ability to produce immunoglobulin (Ig) kappa light chains by targeted deletion of J kappa and C kappa gene segments and the intervening sequences in mouse embryonic stem cells. In wild type mice, approximately 95% of B cells express kappa light chains and only approximately 5% express lambda light chains. Mice heterozygous for the J kappa C kappa deletion have approximately 2-fold more lambda+ B cells than wild-type littermates. Compared with normal mice, homozygous mutants for the J kappa C kappa deletion have about half the number of B cells in both the newly generated and the peripheral B cell compartments, and all of these B cells express lambda light chains in their Ig. Therefore, homozygous mutant mice appear to produce lambda-expressing cells at nearly 10 times the rate observed in normal mice. These findings demonstrate that kappa gene assembly and/or expression is not a prerequisite for lambda gene assembly and expression. Furthermore, there is no detectable rearrangement of 3' kappa RS sequences in lambda+ B cells of the homozygous mutant mice, thus rearrangements of these sequences, per se, is not required for lambda light chain gene assembly. We discuss these findings in the context of their implications for the control of Ig light chain gene rearrangement and potential applications of the mutant animals.     

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.     

Targeted disruption of the porcine immunoglobulin kappa light chain locus

Inactivation of the endogenous pig immunoglobulin (Ig) loci, and replacement with their human counterparts, would produce animals that could alleviate both the supply and specificity issues of therapeutic human polyclonal antibodies (PAbs). Platform genetics are being developed in pigs that have all endogenous Ig loci inactivated and replaced by human counterparts, in order to address this unmet clinical need. This report describes the deletion of the porcine kappa (??) light chain constant (C??) region in pig primary fetal fibroblasts (PPFFs) using gene targeting technology, and the generation of live animals from these cells via somatic cell nuclear transfer (SCNT) cloning. There are only two other targeted loci previously published in swine, and this is the first report of a targeted disruption of an Ig light chain locus in a livestock species. Pigs with one targeted C?? allele (heterozygous knockout or ±) were bred together to generate C?? homozygous knockout (?/?) animals. Peripheral blood mononuclear cells (PBMCs) and mesenteric lymph nodes (MLNs) from C?? ?/? pigs were devoid of ??-containing Igs. Furthermore, there was an increase in lambda (??) light chain expression when compared to that of wild-type littermates (C?? +/+). Targeted inactivation of the Ig heavy chain locus has also been achieved and work is underway to inactivate the pig lambda light chain locus.     

Evolution of immunoglobulin kappa chain variable region genes in vertebrates

The major source of immunoglobulin diversity is variation in DNA sequence among multiple copies of variable region (V) genes of the heavy- and light-chain multigene families. In order to clarify the evolutionary pattern of the multigene family of immunoglobulin light kappa chain V region (V kappa) genes, phylogenetic analyses of V kappa genes from humans and other vertebrate species were conducted. The results obtained indicate that the V kappa genes so far sequenced can be grouped into three major monophyletic clusters, the cartilaginous fish, bony fish and amphibian, and mammalian clusters, and that the cartilaginous fish cluster first separated from the rest of the V kappa genes and then the remaining two clusters diverged. The mammalian V kappa genes can further be divided into 10 V kappa groups, 7 of which are present in the human genome. Human and mouse V kappa genes from different V kappa groups are intermingled rather than clustered on the chromosome, and there are a large number of pseudogenes scattered on the chromosome. This indicates that the chromosomal locations of V kappa genes have been shuffled many times by gene duplication, deletion, and transposition in the evolutionary process and that many genes have become nonfunctional during this process. This mode of evolution is consistent with the model of birth-and-death evolution rather than with the model of concerted evolution. An analysis of duplicate V kappa functional genes and pseudogenes in the human genome has indicated that pseudogenes evolve faster than functional genes but that the rate of nonsynonymous nucleotide substitution in the complementarity-determining regions of V kappa genes has been enhanced by positive Darwinian selection.      

Interallelic and intergenic conversion events could induce differential evolution of the two rabbit immunoglobulin kappa light chain genes.

Contrary to the situation in humans or mice, where the constant region (C) of the Immunoglobulin (Ig) kappa (kappa) light chain is encoded by a single gene, the rabbit possesses two C kappa genes: C kappa 1 and C kappa 2. However, in domestic rabbits, the vast majority of the immunoglobulins have a light chain of the kappa 1 isotype, which is expressed under four complex, highly divergent allelic forms: b4, b5, b6 and b9. In previous papers, we have shown that this high level of divergence was due, at least partly, to conversion events of the kappa 1 by the kappa 2 locus. Up to now, little was known about the evolution of the C kappa 2 gene. Here, we report sequences of the C kappa 2 genes in three different haplotypes, and show that, in contrast to the situation in the kappa 1 locus, the three analysed C kappa 2 alleles are identical (or only differing by one silent substitution). This suggests that intergenic conversion, which introduced most of the divergence in the kappa 1 locus, is not reciprocal and is unidirectional from kappa 2 towards kappa 1. To explain the small number of silent substitutions in the C kappa 2 gene and its remarkable conservation, we propose an extended model of multigenic family evolution, which postulates that gene conversion events occur between linked genes as well as between alleles.     

Notes on individual sequence variation in humans: immunoglobulin kappa light chain.

Little is known concerning the magnitude of variability in the nucleic acid sequence of DNA at the individual level. We have collected a large set of sequence data from the human immunoglobulin kappa light-chain-locus constant region (10,444 bp) and subgroup IV variable region (18,580 bp). For the constant region, absolute conservation of sequence was observed, even in intron and coding-region silent sites, with the exception of one previously defined polymorphic site. For the variable region, 12 heterozygous positions were identified, giving a heterozygosity of 6 x 10(-4) per nucleotide site. The amount of nucleic acid sequence variation differs significantly (chi 2 = 4.88) between these two regions, and the observed variation is two orders of magnitude lower than that reported for two Drosophila melanogaster loci. These data suggest that, for at least some regions of the human genome, nucleic acid sequence may be less variable than previously estimated.     

Plant nonsense suppressor tRNA(Tyr) genes are expressed at very low levels in vitro due to inefficient splicing of the intron-containing pre-tRNAs.

Oligonucleotide-directed mutagenesis was used to generate amber, ochre and opal suppressors from cloned Arabidopsis and Nicotiana tRNA(Tyr) genes. The nonsense suppressor tRNA(Tyr) genes were efficiently transcribed in HeLa and yeast nuclear extracts, however, intron excision from all mutant pre-tRNAs(Tyr) was severely impaired in the homologous wheat germ extract as well as in the yeast in vitro splicing system. The change of one nucleotide in the anticodon of suppressor pre-tRNAs leads to a distortion of the potential intron-anticodon interaction. In order to demonstrate that this caused the reduced splicing efficiency, we created a point mutation in the intron of Arabidopsis tRNA(Tyr) which affected the interaction with the wild-type anticodon. As expected, the resulting pre-tRNA was also inefficiently spliced. Another mutation in the intron, which restored the base-pairing between the amber anticodon and the intron of pre-tRNA(Tyr), resulted in an excellent substrate for wheat germ splicing endonuclease. This type of amber suppressor tRNA(Tyr) gene which yields high levels of mature tRNA(Tyr) should be useful for studying suppression in higher plants.     

Correlation between DNase I hypersensitive sites and putative regulatory sequences in human immunoglobulin genes of the kappa light chain type.

The human lymphoid cell lines Walker and Daudi constitute a particularly suitable system for studies on the chromatin structure of K light chain genes (see preceding paper). The rearranged and non-rearranged alleles of Walker cells were found to be about equally sensitive towards digestion with DNAase I. A DNAase I hypersensitive site was mapped 0.13 kb upstream of the leader segment of the rearranged VK genes; it comprises a region in which promoter-like regulatory elements were discovered recently. Additional hypersensitive sites are located further upstream. A hypersensitive site in the JK-CK intron coincides with a putative tissue specific enhancer element. A hypersensitive region down-stream of CK overlaps with the cleavage/polyadenylation recognition signal which is flanked by sequences related to the above mentioned putative regulatory sequences. The coincidence between DNAase I hypersensitive sites and those sequences may be functionally significant.     

The organization of immunoglobulin variable kappa chain genes on mouse chromosome 6

One mouse with a known recombination (NAK) at the Igk locus on chromosome 6 and two new recombinants [B6.PL (7 NS) and B6.PL (85NS)] were examined using a series of probes, each of which is specific for a set of immunoglobulin (Ig) Vk genes. Under high stringency conditions, each probe detects from 1 to 19 Bam HI restriction endonuclease fragments (REFs) in genomic DNA by Southern transfer hybridization techniques. Analysis of the REF patterns indicate that the NAK recombination event occurred within the variable region of Igk. The REF patterns of the two B6.PL congenic mice provided two additional recombination events which could be examined. Although some of the REFs had shared mobility among the parental strains, at least 1 and up to 13 polymorphic REFs were present for a given probe among the NZB and AKR parental strains. The results from the NAK mouse indicate that at least some members of Vk4, Vk8, Vk10, and Vk21 were on one side of the recombination event linked to the Lyt-2 ^a and Igk-Efl ^a alleles of AKR, while the Vk9, Vk11, and Vk24 REF patterns came from the NZB parental strain linked to the Igk-Ef2 ^b (Vk1) allele. The two B6.PL congenics produced a refined map on the Lyt-2, Lyt-3 side of the Vk region. The B6.PL (85NS) mice retained the Vk21 REF pattern of the Lyt-2 ^a, Lyt-3 ^a donor strain PL/J, while displaying the C57BL/6 REF pattern for the other Vk gene groups tested. The B6.PL (75NS) mice retained the REF patterns of PL/J for Vk21 and Ef-1, indicating a third recombination. This indicates the Vk gene order is (Lyt-2; Vk21); Ef-1; (Vk4; Vk8; Vk10); and (Vk9; Vk11; Vk24; Ef-2).