Richards D A, Blackburn D E, Rosen J M
J Biol Chem. 1981 Jan 10;256(1):533-8.
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.
已确定了三种主要大鼠酪蛋白以及第四种主要乳蛋白α-乳白蛋白的cDNA克隆的详细限制性内切酶图谱。每种乳蛋白基因都表现出独特的限制性内切酶位点。对三种大鼠酪蛋白的限制性内切酶图谱进行比较后发现，它们的基因序列之间没有明显的序列同源性。通过与从经寡聚（dT）-纤维素柱层析分离后部分水解的总聚（A）mRNA制备物合成的3'特异性cDNA探针杂交，确定了每个cDNA基因序列在亲本质粒pBR322中的方向。该技术为确定克隆DNA序列的5'-3'方向提供了一种快速方法。选择了三个方向相同的酪蛋白克隆，并用于异源双链分析，以确定α-、β-和γ-酪蛋白基因内是否存在微小的同源区域。即使在所采用的低严谨度杂交条件下，这些基因之间也未观察到异源双链形成，这表明大鼠酪蛋白基因家族内已发生了相当大的序列分歧。
