Alvin Y. Liu

The primary sequence of rabbit α-globin mRNA

Abstract The rabbit α-globin DNA insertion in the chimeric plasmid pHb72 (Liu et al., 1977) has been sequenced by the method of Maxam and Gilbert (1977). This has enabled us to determine the messenger RNA (mRNA) sequence beginning in the 5′ untranslated region 9 nucleotides before the initiation codon and extending through the first 361 nucleotides of the translated region. The data reported here overlap and are in complete agreement with sequences determined by Baralle (1977) for the 5′ end of the mRNA and by Proudfoot et al. (1977) for the 3′ end. Our sequence is also in agreement with the partial complementary RNA (cRNA) sequencing data which we reported previously (Paddock et al., 1977) and with the published amino acid sequence. Save for three uncertain nucleotides in the 3′ sequence determined by Proudfoot et al. (1977), this work marks the completion of the primary sequence of the rabbit α-globin mRNA. These observations reaffirm the high fidelity with which gene copies can be synthesized in vitro, cloned in a bacterial plasmid and maintained in the host. The general features of the mRNA nucleotide sequence are discussed with particular attention given to the base composition and codon preferences observed and to comparison of this sequence with other completed mRNA gene sequences. A new computer program has been used to search for the most stable base-pairing arrangement of the completed mRNA.

Expression of mouse::human immunoglobulin heavy-chain cDNA in lymphoid cells

A chimeric mouse variable::human constant immunoglobulin heavy-chain gene was expressed in transfected mouse Sp2/0 cells. The chimeric immunoglobulin genes were integrated in tandem in the genome of stably transformed cells. These integrated gene copies were amplified by selection with a second drug marker. The gene amplification led to an increase in the expression of chimeric heavy-chain protein. The level of gene expression appears to be related to the site of integration; a few gene copies in one transfectant can yield as much heavy-chain protein as many copies in a second transfectant. In addition, we found that an adventitious oligo(C) sequence, introduced by our method of gene construction at a site located 8 nt residues downstream from a splice acceptor, can apparently direct splicing towards a cryptic splice acceptor downstream from the oligo(C).