Hybridization properties of immunoglobulin mRNA: Failure to detect covalently associated IgG mRNA transcripts of reiterated and unique mouse DNA

Abstract

Messenger RNAs for antibody heavy (gamma) and light (kappa) chains were isolated from the polysomes of an IgG-producing mouse myeloma cell line. Polysomes engaged in heavy or light chain synthesis were separated by immunoprecipitation using rabbit antibodies specific for the mouse IgG formed. The mRNAs obtained, more than 85% specifying IgG [Legler, M. & Cohen, E. P. (1976) Biochemistry 15, 4390-4399], were used as "probes" in hybridization experiments with sheared mouse liver DNA. To determine whether mRNAs for Ig heavy and light chains contained covalently bound transcripts of unique and reiterated DNA, hybrids were isolated with or without treatment with ribonuclease (RNase) prior to fractionation and the apparent rates of hybridization were compared. A monophasic C(o)t (DNA concentration x incubation time) curve with a C(0)t(1/2) of 4000 moles of nucleotide per liter x sec, corresponding to less than five hybridization sites per haploid genome, was obtained whether or not RNase was used in the isolation protocol. With a similar experimental design, the apparent hybridization rates of heterogeneous nuclear RNA from the same cell source were clearly different. The "stringency" of the reaction was reduced by incubating the hybridization mixtures at lower temperatures in a further attempt to detect a large class of repetitive sequences that would form hybrids with the IgG mRNA used, if such sequences were present. The results, however, were the same; i.e., the apparent rates of hybridization of mRNAs for mouse antibody gamma and kappa chains with sheared mouse liver DNA were essentially the same whether or not RNase was used in the isolation procedure. Reiteration of genes in mouse liver DNA for mouse IgG could not be detected.