No abstract

A rare cDNA coding for most of the alpha subunit of the Torpedo nicotinic acetylcholine receptor has been cloned into bacteria. The use of a mismatched oligonucleotide primer of reverse transcriptase facilitated the design of an efficient, specific probe for recombinant bacteria. DNA sequence analysis has enabled the elucidation of a large part of the polypeptide primary sequence which is discussed in relation to its acetylcholine binding activity and the location of receptor within the plasma membrane. When used as a radioactive probe, the cloned cDNA binds specifically to a single Torpedo mRNA species of about 2350 nucleotides in length but fails to show significant cross-hybridisation with alpha subunit mRNA extracted from cat muscle.

From recently published data on the amino-terminal structures of human and mouse interferons, we have predicted and synthesised an oligonucleotide capable of priming specifically the reverse transcription of human fibroblast interferon mRNA present within a total mRNA population. From these transcripts we determined the sequence of the 5'-terminus of the mRNA and identified a putative pre-peptide signal sequence. This enabled us to predict the sequence of another primer capable of directing the synthesis of interferon double-stranded cDNA corresponding to the entire coding region of the mRNA. Further sequencing studies also enabled us to establish the identity of 47 consecutive amino acids beginning with the methionine residue at the amino-terminus of the mature protein.

Experiments in which immobilised restriction fragments of genomic DNA were hybridised with a cloned human fibroblast interferon cDNA indicate that the homologous chromosomal genes exist in only one basic arrangement. This is in marked contrast to recent studies by Nagata et al. (1) showing that there are at least eight gene arrangements for human leukocyte interferon. Having isolated a chromosomal human fibroblast interferon gene from a gene bank, we conclude from nucleotide sequencing studies that there is a complete absence of introns within the RNA-coding region. In view of a similar observation recently made for a human leukocyte interferon gene (1), it would appear as if interferon genes in general are unlike the vast majority of eukaryote genes in this respect.

Messenger RNA has been isolated from cells of the human myeloma line 266BL which synthesizes IgE of the myeloma ND. A fraction enriched in mRNA for the e heavy chain was copied into DNA and the DNA was cloned in Escherichia coli A chemically synthesized oligonucleotide probe, based on the experimentally determined sequence of the specific message, was used to screen colonies. The largest E chain cDNA cloned, 2.0 kiobases, was characterized by restriction endonuclease mapping and DNA sequence analysis. It appears to encode the complete amino acid sequence ofthe E chain, including a signal peptide at the NH2 terminus as well as untranslated sequences at the 5' and 3' ends of the mRNA. The missing part ofthe previously published amino acid sequence.of the ND E chain was determined from the DNA sequence.The medical importance of IgE, which differs from other immunoglobulins in possessing an E heavy chain, stems from its central role in type 1 immediate hypersensitivity. IgE binds with high affinity to specific receptors on blood basophils, and its association with antigen then triggers an allergic reaction by causing degranulation of the cells (1). Because of the minute concentrations of IgE in normal serum (0.1 Ag/ml, compared with 13 mg/ml for IgG), studies on the purified protein have largely been confined to the secreted products of IgE myelomas. The incidence of IgE myelomas is low, reflecting the corresponding plasma cell population; only three cases (2-4) have been reported so far.For the study ofhuman e gene expression and the molecular and cellular bases of the pathogenicity of IgE, we wished to obtain a cloned DNA sequence that encoded a functional E chain. The usual path to such a clone is by way of isolation and enzymatic copying of mRNA from cells that synthesize the corresponding protein, followed by cDNA cloning. The usual source of immunoglobulin mRNA is an appropriate myeloma or myeloma cell line. In view of the rare occurrence of human IgE myelomas and the fact that human myelomas, in contrast to those of mouse and rat, are notoriously difficult to establish in culture, it was fortunate that Nilsson et aL (5, 6) were able to establish a cell line from the ND myeloma. Its recent adaptation to suspension culture greatly facilitated our endeavors in isolating E chain mRNA. The previously determined sequence of the ND e chain (7) enabled us then to prepare a genetic probe for selecting cDNA clones.The cloned DNA has been used to complete the protein sequence by analysis of the DNA, to correct the earlier variable region sequence and a part ofthe constant region sequence, and to determine the sequence ofthe NH2-terminal signal peptide.This new information has allowed us to identify the D and J elements expressed in the 266BL cell line. We have determined the DNA sequence encoding the 5' and 3' untranslated regions of the mRNA.