Nucleotide sequence analysis of a partially processed polyadenylated precursor RNA transcript shows that the human calcitonin gene in common with the rat calcitonin gene, encodes calcitonin and the calcitonin gene related peptide (CGRP). Using hybridisation probes specific to calcitonin mRNA, intron, coding and non‐coding regions of the CGRP mRNA, we demonstrate by Southern blotting the existence of a second human CGRP gene, and by RNA blotting and S1 mapping, the differential expression of calcitonin and CGRP in medullary thyroid carcinoma and human lung tumour cell‐lines. These studies implicate the requirement for separate post‐transcriptional events in the differential expression of calcitonin and CGRP from a single gene, the preferential use of splice acceptor sites for the synthesis of CGRP mRNA, and post‐transcriptional cleavage modulated by a trans‐acting gene product for the synthesis of calcitonin mRNA. Studies using antisera raised against CGRP and calcitonin, demonstrate elevated circulating levels of plasma CGRP in medullary thyroid carcinoma which do not parallel calcitonin levels, and the presence of CGRP in secretions from lung tumour cell‐lines. These studies indicate that CGRP is a tumour marker of diagnostic and possibly prognostic value in the management of lung and thyroid tumours.

1. Free and membrane-bound polyribosomes were isolated and the associated mRNA species characterized by cell-free protein synthesis, RNA-complexity analysis and polyribosome run-off in vitro. 2. Of the recovered polyribosomal RNA 85% was associated with membrane-bound polyribosomes and contained 87--93% of the total milk-protein mRNA species as assessed by cell-free protein synthesis or RNA-complexity analysis. 3. RNA-complexity analysis showed that the abundant (milk-protein mRNA assumed) species constituted 55% of the post-nuclear poly(A)-containing RNA population, the remainder consisting of a moderately abundant population (18%) and a low abundance population (27%). Calculations suggest that each population contained up to 2, 48 and 5000 different species respectively. 4. RNA-complexity analysis of the free polyribosomal poly(A)-containing RNA demonstrated that all the species in the post-nuclear fraction were present, though in different proportions, the abundant, moderately abundant and low-abundance groups representing 38, 30 and 32% of this population. 5. RNA-complexity analysis of the membrane-bound polyribosomal poly(A)-containing RNA revealed a more limited population, 72% consisting of the abundant (milk-protein mRNA) species, and 28% a population of up to 900 RNA species. 6. Polyribosome run-off confirmed that milk-protein mRNA was associated with the membrane-bound and free polyribosomes, but represented only a small fraction of the total protein synthesized by the latter. 7. Comparative analysis of milk proteins synthesized in mRNA-directed cell-free systems, or by run-off of free and of membrane-bound polyribosomes, is consistent with the interpretation that in vivo the initiation of protein synthesis occurs on free polyribosomes, followed by the attachment of a limited population to the endoplasmic reticulum. After attachment, but before completion of peptide synthesis, the detachable N-terminal peptide sequence of one of these(pre-alpha-lactalbumin) is removed. 8. The results are discussed in terms of the mechanisms involved in the intracellular segregation of mRNA species in the lactating guinea-pig mammary gland.

A recombinant bacteriophage containing the entire alpha-lactalbumin gene was isolated from a human genomic library constructed in bacteriophage lambda L47. Within this recombinant the 2.5 kb alpha-lactalbumin gene is flanked by about 5 kb of sequence on either side. The complete nucleotide sequence of the gene and its immediate flanking sequences were determined and compared with those of the rat alpha-lactalbumin gene. These studies showed that the size, organization and sequence of the exons have been highly conserved, whereas the introns have diverged considerably. In particular, the first intron of the human gene was found to contain an Alu repetitive sequence not present in the rat. A high degree of homology (67%) was also observed in the 5' flanking regions, extending as far as 655 nucleotide residues upstream of the transcriptional initiation site. Comparison of the 5' flanking sequences of these two alpha-lactalbumin genes with those of five casein genes has revealed the presence of a highly conserved region [consensus sequence: RGAAGRAAA(N)TGGACAGAAATCAA(CG)TTTCTA], extending from position -140 to -110 in all seven sequences examined, suggesting a possible regulatory role in the hormonal control or tissue-specific expression of milk protein genes in the mammary gland.

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A complementary DNA (cDNA) plasmid library has been constructed in the plasmid pAT153, using poly(A)-containing RNA isolated from the lactating guinea-pig mammary gland as the starting material. Double stranded cDNA was inserted into the EcoRI site of the plasmid using poly(dA . dT) tails, then transformed into Escherichia coli HB101. From the resulting colonies we have selected and partially characterized plasmids containing cDNA copies of the mRNAs for casein A, casein B, casein C and alpha-lactalbumin. However, the proportion containing casein C cDNA was exceptionally low, and these contained at best 60% of the mRNA sequence.