Molecular analysis of spontaneous somatic mutants

Adetugbo, Kayode; Milstein, C.; Secher, David S.

doi:10.1038/265299a0

Cited by 122 publications

(54 citation statements)

References 38 publications

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“…While many of these spontaneous and mutagen-induced variants have lost the ability to produce immunoglobulin, approximately 40% of the mutagen-induced variants do produce heavy chains that differ structurally from the parental heavy chains (12,13). Milstein and his colleagues have used amino acid and nucleic acid sequence studies to show that frameshift, missense, and nonsense mutations occur spontaneously in their cultured cell lines (8). The genetic instability of immunoglobulin production is not due to a general instability…”

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confidence: 99%

“…In fact, in cultured mouse myeloma cells, the production of immunoglobulins is very unstable (8,9). Spontaneous variants arise at a rate of 10-3/cell per generation, and certain mutagens increase the frequency of variants to as high as 2-6% of surviving clones (10,11).…”

mentioning

confidence: 99%

“…Although this enormous genetic instability in cultured myeloma cells might have suggested a somatic generation of antibody diversity, all of the mutations reported so far (8,12,13) Between 250 and 1000 clones were scored per plate. Subclones and presumptive variants were removed from the agarose and grown up to mass culture as described previously (17).…”

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Antigen-binding mutants of mouse myeloma cells.

Cook¹,

Scharff²

1977

Proc. Natl. Acad. Sci. U.S.A.

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A cultured mouse myeloma cell line, S107, that secretes an IgA phosphocholine-binding immunoglobulin has been cloned in soft agar and overlaid with phosphocholinehemocyanin. Spontaneous mutants that secrete immunoglobulin with a decreased ability to precipitate antigen were detected with this plate assay and occur at a very high frequency. From one such mutant, phenotypic revertants arise spontaneously with a frequency of 0.28-2.8%. This mutant and one of its revertants were studied, and they were found to differ from the parent and from each other serologically and in antigen binding. While it is not yet clear whether these findings bear any relationship to the normal generation of antibody diversity, they do indicate that it is possible to generate antigen binding diversity in somatic cells.

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Antigen-binding mutants of mouse myeloma cells.

Cook¹,

Scharff²

1977

Proc. Natl. Acad. Sci. U.S.A.

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“…Many different deletion mutants of.heavy chains have been described (10)(11)(12)(25)(26)(27)(28)(29). One y H chain mutant, IF2, with an internal deletion of the entire CH1 domain (27,30) (Fig.…”

Section: Discussionmentioning

confidence: 99%

Two alpha heavy chain disease proteins with different genomic deletions demonstrate that nonexpressed alpha heavy chain genes contain methylated bases.

Dackowski¹,

Morrison²

1981

Proc. Natl. Acad. Sci. U.S.A.

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Two independently arising a heavy chain mutants have been found to synthesize heavy chains with CH, deletions ofapproximately equal extent. Both were isolated from heavy chain-producing variants of the mouse myeloma W3129 and demonstrate that it is possible to arrive at the heavy chain disease phenotype by the pathway H + L -e H -+ AH. Analysis of genomic DNA by digestion with restriction endonucleases followed by molecular hybridization showed that one mutant (A37) had a deletion of approximately 0.2 kilobase and the second mutant (A15) had a deletion of approximately 0.5 kilobase. Mouse myeloma cells contain several a chain alleles but only one is expressed; the presence of the deletion in A37 and A15 made it possible to identify the restriction fragments from the expressed allele. Analysis of the fragments produced after cleavage with an isoschizomeric pair of restriction enzymes, Map I and Hpa H, indicated that, in the W3129 cell line and its variants, the unexpressed a alleles contain methylated bases. The influence of methylation on gene expression remains to be elucidated.

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“…Our first structural mutant appeared after a few thousand clones (22), and the final analysis of 7,000 individual clones gave us a pool of mutants which are described in Table 1. We were believed that this elaborate experiment provided the first evidence at the protein and nucleic acid levels of the existence of somatic mutations of mammalian cells (23). Furthermore, the rate at which these mutations occurred suggested an important role in the generation of diversity (24).…”

Section: Spontaneous Somatic Mutants Of a Myeloma Proteinmentioning

confidence: 99%

From the Structure of Antibodies to the Diversification of the Immune Response

Milstein¹

1993

Scand J Immunol

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Cuando se acerca elfin, escribió Cartaphilus, ya no quedan imágenes de1 recuerdo; sólo quedan palabras. Palabras, palabras desplazadas y mutiladas, palabras de otros, fué la pobre limosna que le dejaron las horas y los siglos. J. L. BorgesWhen an animal is infected, either naturally or by experimental injection, with a bacterium, virus, or other foreign body, the animal recognises this as an invader and acts in such a way as to remove or destroy it. There are millions of different chemical structures that the animal has never seen and yet which it is able to recognise in a specific manner. How is this achieved? Scientists have been fascinated by this question for most of this century, and we continue to be fascinated by the intricacies and complexities that still need to be clarified. Even so, looking back over the years since I myself became involved in this problem, progress in the understanding of the process has been phenomenal. Suffice it to remind our younger colleagues that 20 years ago we were still trying to demonstrate that each antibody differed in its primary amino acid sequence. What attracted me to immunology was that the whole thing seemed to revolve around a very simple experiment: take two different antibody molecules and compare their primary sequences. The secret of antibody diversity would emerge from that. Fortunately at the time I was sufficiently ignorant of the subject not to realise how naive I was being.Back in 1962, when I had by accident become the supervisor of Roberto Celis in Argentina, it occurred to me that antibody diversity might arise from the joining by disulphide bridges of a variety of small polypeptides in combinatorial patterns. I don't know whether anybody else had the same idea at that time, but of-all the prevailing theories about antibody diversity that I am aware of, this is one that was widest of the mark. I hold it to my credit that I never put it into print. But it was of great value to me as it provided an intellectual justification to work on disulphide bonds of antibodies. bonds. Each chain has two regions. The variable region differs in structure from one antibody to another and contains the combining site. The antibody combining site is located at the tips of a Yshaped three-dimensional structure. The constant region is invariant within a given class or subclass, and is responsible for effector functions (complement binding, attachment to and transport across membranes etc). The number and position of the interchain disulphide bonds is characteristic for the different classes and subclasses. In this figure, the structure depicted is the mouse myeloma protein MOPC 21 which was the subject of much research in our laboratory.chains joined by disulphide bonds (Fig. 1) had been established (l), and I was eager to accept Dr. Sanger's proposal that I should engage in studies of antibody combining sites. The nature of antibody diversityAt first I looked for differences in fingerprints of digests of iodinated antibodies directed against different antigens. The pattern...

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Molecular analysis of spontaneous somatic mutants

Cited by 122 publications

References 38 publications

Antigen-binding mutants of mouse myeloma cells.

Antigen-binding mutants of mouse myeloma cells.

Two alpha heavy chain disease proteins with different genomic deletions demonstrate that nonexpressed alpha heavy chain genes contain methylated bases.

From the Structure of Antibodies to the Diversification of the Immune Response

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