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Chromosomal location of structural genes encoding murine immunoglobulin lambda light chains. Genetics of murine lambda light chains

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Abstract

To determine the chromosomal localization of murine lambda light (L) chain structural genes, DNA from a panel of 11 mouse x hamster somatic cell hybrids was scored for the presence of sequences homologous to cloned lambda DNA probe molecules. Six of the hybrids had detectable lambda I and lambda II gene sequences. In all six, the full complement of murine sequences was present, and in its germline configuration. The remaining hybrids lacked any detectable murine lambda L chain gene sequences. The only mouse chromosome present in all of the positive hybrids and absent from the negative ones was number 16, allowing the assignment of lambda L chain structural genes to this chromosome. Together with the previous assignments of the kappa L chain genes to chromosome 6 and heavy chain genes to chromosome 12, this finding completes the mapping of Ig structural genes in the mouse at the chromosomal level.

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Chapter
Miss-identification and cross-contamination of cell lines used in research and biotechnology represent widespread problems. Appropriate cell authentication methods should thus be used regularly to avoid invalidating scientific conclusions and to assure the quality of biotechnological products. DNA fingerprinting is often used to establish and verify the identities of various cell lines, but this approach is unsuitable for hybridoma cell lines that are derived from syngeneic animals. Here, we describe the sequencing of antibody variable regions, the only highly diverse region of the hybridoma genome, as a robust and accessible method for hybridoma cell line authentication. The protocol involves RNA isolation, reverse transcription of immunoglobulin variable regions, and amplification of the resulting cDNA using highly degenerate primers, chosen to amplify the majority of the possible variable regions. Depending on the quality of PCR amplification, the PCR products can then be sequenced either directly or after cloning into a plasmid vector. Additionally, we provide an alternative protocol based on rapid amplification of cDNA ends, which can be used to obtain variable region sequences where successful amplification with the degenerate primers is not achieved.
Article
Several inbred or partially inbred mouse strains derived from wild mice of different Mus subgroups were surveyed for expression of lambda 1 light chain. One strain, SPE, expressed little or no lambda 1 chains. Two populations of antibodies were obtained by immunization of SPE mice with lambda 1 light chain isolated from the BALB/c J558 (alpha, lambda 1) myeloma protein. One population of antibodies recognized lambda 1 allotypic determinants located on the C terminal fragment beginning at the residue 176. A second population was directed against epitopes present on both lambda 1 and lambda 3 chains. These determinants, which were detected in all strains tested with the exception of SPE, were located in the V region. This result is in concordance with recent DNA studies showing that the lambda 1 and lambda 3 isotype use the same V lambda 1 genes (Blomberg, B. et al., Proc. Natl. Acad. Sci. USA 1981. 78: 3765). Whether the V epitopes recognized by SPE antibodies are allotypic or isotypic in nature is not certain.
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Tum - comprises a class of genes, mutation of which in P815 tumor cells has led to the acquisition of new cytotoxic T cell-recognized epitopes. The cells carrying the mutant alleles have impaired tumorigenicity compared with their progenitors due to in vivo induction of a cytotoxic T-cell response specific for tum - antigens. Two tum - genes, P91A and P35B, were found to be single copy loci mapping to chromosomes 11 and 15 respectively. A third, P198, was found to map to chromosome 7 and to be a member of a small gene family with other members on chromosomes 13, 14, and 15. Multiple P198-related sequences were found in other mammalian species suggesting the P198 related gene family is a general feature of mammalian genomes.
Article
Some of the homeotic genes of Drosophila, involved in the control of segmental development, form a diverged multigene family. A conserved DNA sequence common to these genes has been used to isolate a clone (Mo-10) from the mouse genome which contains a sequence coding for a protein domain that is homologous to the domain conserved in the Drosophila homeotic genes. By structural analogy, this sequence may be involved in the control of metameric pattern formation in the mouse. Mo-10 has been mapped to the proximal portion of mouse chromosome 6, and its position in relationship to genes known to influence mouse morphogenesis is discussed.
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Mouse-hamster somatic cell hybrids containing a variable number of mouse chromosomes have been used in experiments to determine which mouse chromosome carries the immunoglobulin kappa light chain genes. It has been shown by nucleic acid hybridization that the kappa constant gene and the genes for at least one variable region subgroup are on mouse chromosome 6. This somatic cell genetic mapping procedure appears to be general and can be applied to any expressed or silent gene for which an appropriate nucleic acid probe exists.
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The gene for the receptor for ecotropic murine leukemia virus (Rev) has been assigned to mouse chromosome 5. This determination was made possible by an analysis of somatic cell hybrids between mouse and Chinese hamster cells. The parents of these hybrids were A/HeJ or Mus poschiavinus peritoneal exudate cells or BALB/c primary embryo fibroblasts and E36, a Chinese hamster lung fibroblast deficient in hypoxanthine guanine phosphoribosyltransferase. Segregation of mouse chromosomes in these hybrids was analyzed by chromosome banding and isozyme expression. Cells were tested for their ability to absorb and replicate vesicular stomatitis virus (murine leukemia virus [MuLV]) pseudotype particles and ecotropic MuLV as measured by the XC test. The presence of chromosome 5 was essential for receptor expression as determined by three statistical procedures. Segregation of the receptor for ecotropic murine leukemia virus was also followed in two series of subclones. In both, receptor expression was syntenic with phosphoglucomutase-1, an isozyme which has been mapped to mouse chromosome 5.
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Metaphase chromosomes isolated from a cell line carrying the thymidine kinase (TK) gene of herpes simplex virus type I were used to transform the TK-deficient cell line LMTK- to the TK+ phenotype. Four independent transformants were isolated, all of which expressed virus-specific TK. Each of the four transformant cell lines initially became TK- at a rate of 12% per day. All four transformants possessed multiple copies of the TK gene and in one of the four a rearrangement occurred adjacent to the TK sequences. Stable TK+ derivatives of each line, isolated after prolonged cultivation, retained fewer copies of the TK gene than did their unstable parents. The transferred chromosomal fragment was larger than 17 kilobases in each line.
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We describe a technique for transferring electrophoretically separated bands of double-stranded DNA from agarose gels to diazobenzyloxymethyl-paper. Controlled cleavage of the DNA in situ by sequential treatment with dilute acid, which causes partial depurination, and dilute alkali, which causes cleavage and separation of the strands, allows the DNA to leave the gel rapidly and completely, with an efficiency independent of its size. Covalent attachment of DNA to paper prevents losses during subsequent hybridization and washing steps and allows a single paper to be reused many times. Ten percent dextran sulfate, originally found to accelerate DNA hybridization in solution by about 10-fold [J.G. Wetmur (1975) Biopolymers 14, 2517-2524], accelerates the rate of hybridization of randomly cleaved double-stranded DNA probes to immobilized nucleic acids by as much as 100-fold, without increasing the background significantly.
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The sequence of 72 base pairs of the rightward operator (O-R) of bacteriophage lambda is presented as determined with simple and rapid methods for direct DNA sequencing. The sequence of an operator mutant is also described. The methods are of general use in sequencing DNA fragments with unique 5' ends up to 50 base pairs in length. Previous experiments have shown that this operator contains multiple sites recognized by the lambda phage repressor. We believe we have identified three of these sites.
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Amino-acid sequences of the variable regions of three lambda chains produced by plasmacytomas of BALB/c mice are compared. Two are almost certainly identical and one differs from these by three amino acids. These findings extend our earlier conclusion on the relative uniformity of sequences in this type of immunoglobulin light chain. With amino-acid sequence data on two additional lambda chains, eight mouse lambda chains studied to date are indistinguishable and four probably differ from these by one, two, or three amino acids.
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Analysis of backcross mice carrying the Harwell translocation T(5;12)31H has led to the definitive localization of the immunoglobulin heavy chain gene cluster. Both Igh-1 and Pre-1 loci were found to segregate in tight linkage with the chromosomal markers 5(12) and 12(5), which define the balanced translocation T(5;12)31H. Additional data establish the location of these genes at the telomeric end of chromosome 12. That both loci are proximal to the chromosomal breakpoint in band 12F1 is shown by the phenotypes of segregants aneuploid for the presence or absence of the small marker 5(12). The order of loci inferred from a single recovered recombinant is: centromere-Igh-1-Pre-1-T(5;12)31H.
Article
The heterogeneity of immunoglobulins of a single vertebrate species is because of the fact that there are a number of genes in the germ line that dictate the structure, or control the expression, of different immunoglobulin polypeptide chains. Genetic polymorphisms of many of these genes have been discovered by serologic and chemical methods. The genetic markers on different portions of various polypeptide chains of the immunoglobulins have provided powerful tools for the study of the genetic basis of antibody structure. The allotypes of human immunoglobulins were discovered during an investigation into the anti-immunoglobulins that were present in sera of patients with hypogammaglobulinemia. The modern era of investigation of rabbit allotypes began with the discovery of isoprecipitins in the sera of rabbits injected with specific precipitates formed between antiserum of a second rabbit and corresponding antigen. Since these initial discoveries, investigations of allotypes have contributed to the understanding of immunoglobulin structure and genetics and to the concepts of the complexities of cellular interactions in development, immunity, and specific immune responses. This chapter reviews the knowledge of the genetically different forms of immunoglobulins in man, mouse, and rabbit.
Article
The chromosomal locations of the structural genes coding for the constant portions of mouse heavy (H) and light chain immunoglobulins were studied by molecular hybridization techniques. Complementary DNA probes containing the constant-region sequences of kappa and lambdaI light chain and alpha, gamma2b, and mu heavy chain mRNAs were annealed to a large excess of DNA from a series of eight mouse-human hybrid cell lines that are deficient for various mouse chromosomes. The lines were scored as positive when a high proportion of a probe annealed and negative when an insignificant proportion annealed. Some lines were clearly negative for H and lambda and clearly positive for kappa. Others were positive or intermediate for lambda, positive for kappa and negative for H. Still others, including a line that was selected for the absence of the mouse X chromosome, were positive for all immunoglobulin species. These results demonstrate that the Clambda, Ckappa, and CH genes are located on different autosomes in the mouse. In contrast, the three heavy-chain families exhibited consistently uniform hybridization results, suggesting that the genes for Calpha, Cgamma, and Cmu are located on the same chromosome. A comparison of karyotypic data with hybridization data has limited the possible locations of the Ig genes to only a few chromosomes.
Article
The entire nucleotide sequence of a 1.7-kilobase embryonic DNA fragment containing five joining (J) DNA segments for mouse immunoglobulin kappa chain gene has been determined. Each J DNA segment can encode amino acid residues 96--108. Comparison of one of the five J DNA sequences with those of an embryonic variable (V) gene and a complete kappa chain gene permitted localisation of a precise recombination site. The 5'-flanking regions of J DNA segments could form an inverted stem structure with the 3'-non-coding region of embryonic V genes. This hypothetical structure and gel-blotting analysis of total embryo and myeloma DNA suggest that the somatic recombination may be accompanied by excision of an entire DNA segment between a V gene and a J DNA segment. Antibody diversity may in part be generated by modulation of the precise recombination sites.
Article
Transfection experiments with HSV 1 in which one uses herpes simplex virus (HSV) thymidine kinase (TK) as a selectable prototrophic marker yield two classes of transformed cells: stable and unstable. In this report, we test the hypothesis that the stability phenotype can be explained by virus genome integration into a recipient cell chromosome. The method of analysis is by means of somatic cell genetics. We have isolated a series of microcell hybrids between a TK- Chinese hamster cell line and a transformed mouse cell line expressing the TK encoded by HSV 1. Several of the hybrid lines contain a single murine chromosome and express only the viral TK. Karyotypic analysis of these hybrids and of TK- derivatives generated by BrdUrd counterselection reveals that the TK+ phenotype is correlated with the presence of the terminal portion of the long arm of a specific murine chromosome. Results of extensive isozyme analyses of the hybrids and their TK- segregants fully corroborate the karyologic data. The results are consistent with the hypothesis that the viral tk gene is covalently integrated into this chromosomal region which itself does not appear to carry the endogenous murine tk locus. Other more complicated models are discussed. Our findings also show that somatic cell genetics can be used to localize viral integration sites in host chromosomes with high resolution.
Article
Trispecific microcell hybrids were prepared by transferring limited numbers of chromosomes from a human/mouse gene-transfer cell line to a Chinese hamster recipient line. The donor cells employed were murine L-cells that stably expressed the human form of the enzyme hypoxanthine phosphoribosyltransferase. Karyotypic, zymographic, and back-selection tests of the resulting human/mouse/Chinese hamster microcell hybrids provided strong genetic evidence for a stable association of the human transgenome with host murine chromosomes in stable gene-transfer cell lines. This association, which may represent physical integration of the transgenome into the host cell genome, occurred at multiple chromosomal sites.
Article
The inbred and congenic strain distribution of the I(H)-peptide marker in the variable region of mouse immunoglobulin light chains has been compared with other known genetic markers. A positive correlation was noted between the I(H)-peptide marker and expression of the Ly-3.1 thymocyte cell surface antigen. This suggests that the locus responsible for I(H)-peptide expression is genetically linked to the Ly-2 and Ly-3 loci in linkage group XI on chromsome 6 of the mouse.
Article
This communication describes the isolation and characterization of tryptic and thermolytic peptides and cyanogen bromide fragments of the light chain of protein 315, a mouse IgA myeloma protein with anti-hapten activity. Some peptides were aligned by analogy with amino acid sequences of closely related immunoglobulin chains; the results provide, with the exception of eight residues, a complete covalent structure. The sequence suggests that this chain (L315) represents a new type of mouse light chain, more like λ than k chains. It is proposed that the more frequently encountered type of λ chains be designated as λ1 and L315 as λ2.
Article
To determine the chromosomal location of mouse immunoglobulin heavy chain structural genes unambiguously, a panel of somatic cell hybrids was scored for the presence of DNA sequences homologous to gamma 2b-, mu-, and alpha-heavy chain-constant region DNA probe molecules. The hybrids, formed between mouse and hamster cells, contained various combinations of mouse chromosomes plus a full set of hamster chromosomes. Hybrids that retained mouse chromosome 12 reacted with the probes, whereas hybrids that had lost the chromosome, or its distal half, failed to react. These results indicate that structural genes for the gamma 2b-, mu-, and alpha-heavy chain-constant regions map to the distal half of this chromosome.
Structure of immunoglobulins
  • I Gaily
I. Gaily, J. A. 1973. Structure of immunoglobulins. In The Antigens. M. Sela, editor. Academic Press, Inc., New York. 161.
Immunoglobulin aIIotypes
  • R Mage
  • R Lieberman
  • M Potter
  • W D Terry
Mage, R., R. Lieberman, M. Potter, and W. D. Terry. 1973. Immunoglobulin aIIotypes. In The Antigens. M. Sela, editor. Academic Press, Inc., New York. 299.
Sequences at the somatic recombination sites of immunoglobulin light-chain genes
  • H Sakano
  • K Hfippi
  • G Heinrich
  • S Tonegawa
Sakano, H., K. Hfippi, G. Heinrich, and S. Tonegawa. 1979. Sequences at the somatic recombination sites of immunoglobulin light-chain genes. Nature (Lond.). 280:288.