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D E Blackburn

Publications and source records attributed to D E Blackburn.

2 recordsLinked to original sources

Rat beta casein cDNA: sequence analysis and evolutionary comparisons.

The complete sequence of a 1072 nucleotide rat beta-casein cDNA insertion in the hybrid plasmid pC beta 23 has been determined. Primer extension was employed to determine the sequence of an additional 82 5'-terminal nucleotides in beta-casein mRNA. Rat beta-casein mRNA consists of a 696 nucleotide coding region, flanked by 52 nucleotide 5' and 406 nucleotide 3' noncoding regions, including a 40 nucleotide poly(A) tail. The derived 216 amino acid sequence of rat beta-casein was compared to the previously determined sequences of beta-caseins from several other species. Approximately 38% of the amino acids have been conserved among the rat, ovine, bovine and human sequences and these conserved amino acids occurred in clusters throughout the protein. One such cluster containing the majority of the potential casein phosphorylation sites was located near the amino terminus. Contrary to the considerable divergence observed for the processed beta-casein, 14 of 15 amino acids in the signal peptide sequence of the precasein were identical between the rat and ovine caseins.

Amino Acid Sequence

Restriction enzyme mapping and heteroduplex analysis of the rat milk protein cDNA clones.

Detailed restriction enzyme maps have been determined for the three major rat casein and the fourth principal milk protein, alpha-lactalbumin, cDNA clones. Each of the milk protein genes exhibited unique and characteristic restriction enzyme sites. A comparison of the restriction enzyme maps of the three rat caseins revealed no apparent sequence homology among their gene sequences. The orientation of each cDNA gene sequence within the parent plasmid, pBR322, was determined by hybridization with a 3' specific cDNA probe synthesized from a partially hydrolyzed total poly(A) mRNA preparation following isolation by chromatography on oligo(dT)-cellulose. This technique provided a rapid procedure for determining the 5'-3' orientation of the cloned DNA sequences. Three casein clones were selected, which were in the same orientation, and were employed for a heteroduplex analysis to determine whether minor regions of homology existed within the alpha-, beta-, and gamma-casein genes. No heteroduplex formation was observed among these genes even under the low stringency conditions of hybridization employed, suggesting that considerable sequence divergence has occurred within the rat casein gene family.

Animals