Nucleotide sequence of the 3'-nontranslatable region of mRNA of cow. cap alpha. /sub s/sub 1//-casein

In this paper, the authors make a determination of the nucleotide sequence of the 3'-nontranslatable region of cow ..cap alpha../sub s/sub 1//-casein mRNA and an analysis of the degree of homology with the corresponding region in rat mRNA. In subsequent analysis, the authors established that in the cloned sequence of cDNA (740 nucleotide pairs) there are no recognition sites for the restriction endonucleases MspI and PstI, and there is one site for the restriction endonuclease EcoRI. On the basis of restriction analysis and blot hybridization with /sup 32/P-labeled poly(T), the authors determined the orientation of cDNA in the DNA of the plasmid pK cas ..cap alpha../sub s/sub 1//-17. These data were used in selecting approaches to the determination of the nucleotide sequence. Labeled DNA fragments were obtained by hydrolysis of the plasmid DNA with the restriction endonuclease EcoRI, followed by introduction of the label at the ends with the aid of E. coli DNA polymerase I (large fragment) in the presence of (/sup 32/P)-dATP and three unlabeled dNTP. After supplementary hydrolysis of DNA with the restriction endonuclease PstI, the mixture was fractionated by electrophoresis in 5% polyacrylamide gel, and the labeled fragments corresponding to cDNA 9450 and 300 n.p. weremore » extracted.« less