The extent of diversity of anti‐hapten antibodies in inbred mice: anti‐NIP (4‐hydroxy‐5‐iodo‐3‐nitro‐phenacetyl) antibodies in CBA/H mice

Kreth, H. W.; Williamson, Alan R.

doi:10.1002/eji.1830030306

Cited by 118 publications

(45 citation statements)

References 14 publications

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“…The estimate of purity made in this way is in excellent agreement with the estimate from the fingerprint studies. Half maximal competition was reached at an RNA to DNA ratio of 1 Using a mouse K-chain mRNA preparation of unknown purity, Delovitch and Baglioni (24) observed a biphasic Cot curve which, to a certain extent, is similar to ours. They also ignored the existence of the external section of the mRNA and assumed that the reiterated fraction represented "subclass" specific sequences.…”

Section: Hybridization Kineticssupporting

confidence: 80%

See 1 more Smart Citation

Evidence for Somatic Generation of Antibody Diversity

Tonegawa¹,

Steinberg²,

Dube

et al. 1974

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

114

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RNA preparations containing 70-80% mouse K-chain mRNA have been prepared. The remainder consists of many RNA species, each of which represents a small fraction of the total RNA. The K-chain mRNA preparation hybridizes with mouse liver DNA with biphasic kinetics, indicating that it consists of two fractions -"unique" and "reiterated." Competition hybridization experiments show that the homology among the unique fractions from different mRNAs is the same as the homology among the amino acid sequences of the corresponding K-chains. Hence, in addition to the C-region (constantregion) sequences, (most of) the V-region (variableregion) sequences are also derived from unique germ line genes. The reiterated fractions from different K-chain mRNAs show essentially complete homology with each other. This fraction seems to consist mostly of sequences which do not code for amino-acid sequences of the secreted polypeptide chain, i.e., the "external" section of the mRNA molecule. It is concluded that the number of germ line genes is too small to account for the observed diversity of antibody molecules.One of the most fascinating problems of immunology is posed by the enormous diversity of the antibody molecules that one animal is able to synthesize. One inbred strain of mice has been shown to be capable of producing eight thousand different antibody molecules against the hapten NIP (4-hydroxy-5-iodo-3-nitrophenacetyl) (1). Another inbred mouse strain was shown to produce many different antibody molecules against DNP (dinitrophenyl), of which more than 500 crossreact strongly with TNP (trinitrophenyl) (2). The occurrence of species-specific residues in the V-regions of antibody polypeptide chains and the mendelian inheritance of rabbit a allotypes point against a germ line theory (3). Nevertheless, controversial ideas of evolutionary versus somatic generation of antibody diversity have continued to coexist (3, 4). The direct approach to solving this controversy is to count the number of antibody V-genes present in the DNA of one cell. To this end, we have analysed the kinetics of hybridization of a mouse K-chain mRNA to mouse liver DNA. For the analysis to be meaningful, three points are essential:1. The physical purity of the K-chain mRNA preparation must be known; that is, the amount and nature of contaminants must be determined.Abbreviations: Cot, moles of deoxyribonucleotide,/liter of incubation X see; SSC, standard saline-citrate solution (0.15 M sodium chloride-0.015 M sodium citrate, pH 7); 2 X SSC means that the concentration of the solution used is two times that of the standard saline-citrate solution; V-region, variable region; Cregion, constant region. 40272. Since K-chain mRNA contains both a unique and a reiterated fraction (5, 6), it is necessary to assign the V-region sequences to one of these fractions.3. The proportion of all possible V-genes which would have cross-hybridized with V-region sequences of the particular K-chain mRNA used, must be determined.We have recently reported a preliminary account ...

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Section: Hybridization Kineticssupporting

confidence: 80%

“…One inbred strain of mice has been shown to be capable of producing eight thousand different antibody molecules against the hapten NIP (4-hydroxy-5-iodo-3-nitrophenacetyl) (1). Another inbred mouse strain was shown to produce many different antibody molecules against DNP (dinitrophenyl), of which more than 500 crossreact strongly with TNP (trinitrophenyl) (2).…”

mentioning

confidence: 99%

Evidence for Somatic Generation of Antibody Diversity

Tonegawa¹,

Steinberg²,

Dube

et al. 1974

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

114

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“…Although responses in mice to certain antigenic determinants may invariably include a "dominant" clonotype (15-17), a study by Kreth and Williamson has indicated that the immune response of CBA/H mice to the 4-hydroxy-5-iodo-3-nitrophenylacetyl (NIP) determinant may involve as many as 3,000-30,000 distinct clonotypes (18) . Given such an enormous array of clonotypes in the adult mouse potentially available for responsiveness to determinants such as NIP and probably to DNP as well, the analysis of the generation of the specificity repertoire to these determinants is extremely hampered .…”

Section: Discussionmentioning

confidence: 99%

The characterization fo the B-cell repertoire specific for the 2,4-dinitrophenyl and 2,4,6-trinitrophenyl determinants in neonatal BALB/c mice.

Klinman¹,

Press²

1975

The Journal of Experimental Medicine

109

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The process by which an individual's repertoire of unique antibody-forming cell precursor (B-cell)' specificities (clonotypes) is acquired has been the subject of extensive investigation and controversy over the past several years (1-3) . To help clarify the mechanism by which diversification of B-cell clonotypes might occur, this laboratory has conducted an investigation of the B-cell clonotypes available during the early stages of neonatal development, i .e ., at an important period in the initial development of the specificity repertoire .Previous reports from this laboratory have demonstrated that the adoptive transfer of neonatal spleen cells to an adult, carrier-primed environment maximizes the responsiveness of neonatal B cells (4-6) . The principle rationale for this approach-that neonatal B cells are indeed immunologically competent when provided with ancillary mechanisms by the adult host environment-has been verified by other investigators (7,8) . Previous analyses from this laboratory have also shown the similarity of neonatal and adult primary B cells, in terms of the kinetics and antigen dose dependence of the antibody response (4), and the relative ease of hapten inhibition of primary B-cell stimulation (6,9,10) . Recently, an analysis of the frequency of neonatal B-cell precursors specific for the haptenic determinants 2,4-dinitrophenyl (DNP) ; 2,4,6-trinitrophenyl (TNP) ; and fluorescein (FL) has demonstrated that a disparity exists in the rate of development of FL-specific precursor cells, as compared to DNP-or TNP-specific cells (5) . Thus, at an early stage in the generation of specificity, when the total number of available specificities is limited, significant disparities may exist due to potential "all or none" expressions .This report extends these observations by an analysis of the clonotypes available in neonatal BALB/c mice which are specific for the DNP and TNP haptenic *

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“…Recent experiments measuring the number of nitro-iodohydroxy-phenacetyl-binding antibodies which can be made in CBA/H mice indicated a minimum number of 70 VH-genes and 70 VL-genes are required if all of the information is carried in the germ-line (19). This number would be comfortably contained in a VH-pOO1 of 5000 genes.…”

Section: Discussionmentioning

confidence: 99%

Germ Line Basis for Antibody Diversity: Immunoglobulin V _H - and C _H -gene Frequencies Measured by DNA·RNA Hybridization

Premkumar¹,

Shoyab²,

Williamson³

1974

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

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The reiteration frequency for mouse im- Immunoglobulin heavy (H) or light (L) chain structure can be explained by assuming that each chain is encoded by two genes, one variable (V)-and one constant (C)-gene (1). Heavy chains of all classes share the sme VH-gene pool and different CH-genes can be linked to the same VH-gene. One implication of this two gene-one polypeptide chain hypothesis is that there is no necessity for an equal number of V-and C-genes in the germ line. This satisfies the genetic evidence for a single C-gene for each constant region while permitting multiple V-genes as implied by amino-acid sequence data. That data points to a minimum essential number of V-genes which could account for the extent of antibody diversity only if there is a superimposed somatic generation of diversity (2-5). Alternatively a larger number of V-genes could be present in the germ-line and this could easily be a sufficient number to code for the total repetoire of antibodies without invoking diversification (1, 6, 7). A direct estimate of the reiteration frequency of a given gene can be made by hybridization of a suitable radioactive probe, either RNA or DNA, in the presence of a vast excess of DNA (8). The probe, either mRNA or a reverse transcript thereof, must be labeled at very high specific activity so that the vast excess of DNA can be achieved.We have used as a probe the mRNA for the a-chain of mouse myeloma protein 315. The mRNA was labeled in 315 cells grown in tissue culture and was isolated by virtue of its specific interaction with immunoglobulin (refs 9 and 10, Premkumar, Stevens, and Williamson, in preparation (11), and the total RNA was isolated from the cytoplasmic fraction by the phenol-chloroform method of Singer and Penman (12). After phenol extraction, the RNA was precipitated with 2 volumes of ethanol overnight at -20°.The RNA precipitates were collected by centrifugation, dissolved in water, and fractionated into poly(A) containing RNA and nonpoly(A) RNA by the method of Schutz, Beato, and Feigelson (13). The poly(A) containing RNA fraction was precipitated with 3 volumes of ethanol overnight at -20°.The precipitate was collected by centrifugation, dissolved in 0.5 ml of solution A [0.5 mM MgCl2-75 mM NaCl-20 mM phosphate buffer (pH 7.6)], 50,g of 315 protein, purified by affinity chromatography on DNP-Sepharose column (14), in Solution A was added and the mixture incubated on ice for 3 min. Anti-315 antibody, purified on DEAE-Sephadex, was added at equivalence and the mixture was incubated on ice for a further 5 min (9). The precipitates formed were collected by centrifugation (3000 X g for 10 min) and washed three times with ice cold solution A. The precipitates were dissolved in 0.5 ml of 0.5% sodium dodecyl sulfate (SDS) solution and then diluted to 5 ml with RNA extraction buffer (0.1 M sodium acetate-0.2 M NaCl-1 mM EDTA-0.2% SDS), pH brought to 5.0 with acetic acid, and shaken with an equal volume of phenol-chloroform (3:1) mixture at 450 for 3 min.The mixture was then chilled, cen...

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The extent of diversity of anti‐hapten antibodies in inbred mice: anti‐NIP (4‐hydroxy‐5‐iodo‐3‐nitro‐phenacetyl) antibodies in CBA/H mice

Cited by 118 publications

References 14 publications

Evidence for Somatic Generation of Antibody Diversity

Evidence for Somatic Generation of Antibody Diversity

The characterization fo the B-cell repertoire specific for the 2,4-dinitrophenyl and 2,4,6-trinitrophenyl determinants in neonatal BALB/c mice.

Germ Line Basis for Antibody Diversity: Immunoglobulin V _H - and C _H -gene Frequencies Measured by DNA·RNA Hybridization

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The extent of diversity of anti‐hapten antibodies in inbred mice: anti‐NIP (4‐hydroxy‐5‐iodo‐3‐nitro‐phenacetyl) antibodies in CBA/H mice

Cited by 118 publications

References 14 publications

Evidence for Somatic Generation of Antibody Diversity

Evidence for Somatic Generation of Antibody Diversity

The characterization fo the B-cell repertoire specific for the 2,4-dinitrophenyl and 2,4,6-trinitrophenyl determinants in neonatal BALB/c mice.

Germ Line Basis for Antibody Diversity: Immunoglobulin V H - and C H -gene Frequencies Measured by DNA·RNA Hybridization

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Product

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Germ Line Basis for Antibody Diversity: Immunoglobulin V _H - and C _H -gene Frequencies Measured by DNA·RNA Hybridization