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

Abstract

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...

The Organization and Diversity of Immunoglobulin Genes

“…On the basis of these expectations, it is quite clear that we could begin to distinguish between these models were we able to quantitate the immunoglobulin constant and variable region gene sequences. Our studies and those of a number of other laboratories have, in fact, been directed towards this goal (1,(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23). This paper reviews the progress we have made.…”

“…The hybridization procedure and SI nuclease assays were as described (38), and were performed at DNA concentrations and salt concentrations appropriate for each portion of the kinetic curve (see below). The Cot values on the abscissa are those that would obtain at 0.18 M Na+ (26) (11,20,22,23). The interpretation of data obtained from such studies is necessarily limited by uncertainties regarding the purity of the hybridization probe and/or the portion of the hybridization curve representing the V-and Cregions.…”

Size differences among immunoglobulin heavy chains from phosphorylcholine-binding proteins.

“…The mechanism(s) of generation of immunoglobulin diversity has long been a subject of major controversy. Attempts have been made to approach this problem by primary sequence analysis (1)(2)(3)(4), studies of the inheritance of serological markers (5-7), and more recently nucleic acid hybridization (8)(9)(10)(11)(12)(13)(14). The mouse plasmacytoma system provides a unique opportunity to observe the variation in primary structure among proteins with similar antigen binding specificity.…”

No detectable reiteration of genes coding for mouse MOPC 41 immunoglobulin light-chain mRNA.

“…copies (37 (Fig. 3), analogous to that observed with 14S mRNA, has been reported for various preparations of L-chain mRNA (11)(12)(13)(14)(15), as well as for fractions containing Ig heavy chain mRNA (16,17). With a single exception (16), this rapidly hybridizing RNA corresponds to DNA sequences reiterated 200 to 300 times.…”