Nucleic Acids Research

The nucleic acid sequence of the preprolnsulln cDNA of carp (Cyprinus carpio), cloned in the Pstl-site of pBR322 (i), has been determined. The sequenced insert of 439 bp includes the complete coding Information for carp preproinsulin (108 amlno acids), 10 nucleotides of the 5'-and 105 nucleotides of the 3'nontranslated regions. The nucleotlde sequence confirms the previously established amino acid sequence of carp insulin (2) and determines those of the signal (21 aa ) and C-peptlde (35 aa 1). The observed shortness of the signal peptide of carp preproinsulin and the N-terminal addition of 2 amino acids to the carp Insulin B-chain suggest that the cleavage site of the signal peptldase has moved. Calculations based on the comparison of known preproinsulin cONA sequences showed that the evolutionary distance between fresh water and salt water teleostians is not smaller than between man and chicken. INTRODUCTION Although insulin has been studied intensively for a long time, a new level in this research has been reached with the modern recombinant ONA technology and nucleic acid sequencing methods, which made it possible to isolate and sequence the mRNAs and chromosomal genes coding for single proteins. Thus the cloning of cONA copies of the mRNA encoding human (3,4), rat (5,6,7), carp (1), anglerflsh (8), hagfish (9) and salmon (10,11) preproinsulin has been reported. In addition genomic fragments containing rat Insulin genes I and II (4,12,13) the human Insulin gene (4,14) and the chicken insulin gene (15) were Isolated and sequencoti. Continuing our work on carp preproinsulin we report here the nucleotide sequence for carp preproinsulin cDNA and the amino 1) aa = amino acids © IRL Press Limited, Oxford, England. 4 5 4 1 Nucleic Acids Research acid sequence derived from it. To verify the nucleic acid sequencing data the carp insulin amino acid sequence has been estimated independently and already reported (2). The knowledge of the preproinsulin sequence of carp (Cyprinus carpio), a fresh water fish, allows a valuable extension of the comparisons of evolutionary distant vertebrate insulin genes. Our data give new insights Into structural similarities and differences in relation to the evolutionary process. MATERIAL AND METHODS a) Enzymes Restriction enzymes were either commercial products from Boehringer Mannheim XPst *» Bam HI, Hind IX, Hpa II, Taq I) and New England Biolabs (Ava II) or enzymes prepared in the lab of Dr. M. Hartmann (ZIMET, Oena, GDR) : Pst I, Bam HI, Hinc II and Bsp RI. Calf intestine alkaline phosphatase and polynucleotide kinase were obtained from Boehringer Mannheim. In addition some polynucleotlde kinase samples were gifts of Dr. P. Venetianer (BRC, Szeged, Hungary). b) Techniques: Plasmid DNA isolation, restriction -, nucleic acid sequence and computer analysis Plasmid DNA was isolated by the cleared lysate method (16) and further purified by gel filtration on sepharose 4B and by CsCl-density gradient centrlfugation (17). Restriction endonucleases were used according to the manufacturers recommendations. DNA sequence analysis was performed using the chemical method developed by Maxam and Gilbert (18). For the comparison of sequences, prediction of restriction sites and homology considoratlons, computer programs developed by Dr. 0. Winkler for a HP 9845 have been used. RESULTS As described in our previous paper (1) the cloning of ds cDNA, synthesized at poly A_mRNA from carp islets was done in the unique Pst I-slte of pBR322 using the dG/dC tailing technique. With the hybrid arrested translation system the clone pLCR665 was selected as most likely carrying the Information for carp