Alevy M C, Tsai M J, O'Malley B W
Biochemistry. 1984 May 8;23(10):2309-14. doi: 10.1021/bi00305a034.
We have cloned a 36-kilobase segment of chicken DNA containing the gene coding for glyceraldehyde-3-phosphate dehydrogenase [GAPDH (EC 1.2.1.12)], a glycolytic enzyme which is expressed constitutively in all cell types. Using defined segments of this cloned DNA as probes, we have determined the DNase I sensitive domain of the GAPDH natural gene in the hen oviduct. When nuclei isolated from hen oviduct were treated with DNase I under conditions known to preferentially degrade actively transcribed genes (i.e., 15-20% of the DNA rendered perchloric acid soluble), a region of approximately 12 kilobase(s) (kb) containing the GAPDH coding sequences and flanking DNA was found to be highly susceptible to digestion by DNase I. Approximately 4 kb downstream from the end of the coding sequences, there was an abrupt transition from the DNase I sensitive or "open" configuration to the resistant or "closed" configuration. The chromatin then remained in a closed conformation for at least 10 kb further downstream. On the 5' side of the gene, the transition from a sensitive to a resistant configuration was located about 4 kb upstream from the gene. In addition, we have localized two repeated sequences in the area of DNA that was cloned. One of these is of the CR1 family of middle repetitive elements. It is located about 18 kb 3' to the gene and as such lies well outside of the DNase I sensitive region which encompasses GAPDH. The other repetitive element is of an uncharacterized family. It is located upstream from the gene and appears to be within a region of transition from the DNase I sensitive to resistant states.
我们克隆了一段36千碱基的鸡DNA片段，其中包含编码甘油醛-3-磷酸脱氢酶[GAPDH（EC 1.2.1.12）]的基因，该糖酵解酶在所有细胞类型中均组成性表达。使用该克隆DNA的特定片段作为探针，我们确定了母鸡输卵管中GAPDH天然基因的DNase I敏感区域。当在已知优先降解活跃转录基因的条件下（即15 - 20%的DNA可被高氯酸溶解）用DNase I处理从母鸡输卵管分离的细胞核时，发现一个约12千碱基（kb）的区域，包含GAPDH编码序列和侧翼DNA，对DNase I消化高度敏感。在编码序列末端下游约4 kb处，从DNase I敏感或“开放”构型到抗性或“封闭”构型有一个突然转变。染色质在下游至少10 kb内保持封闭构象。在基因的5'端，从敏感构型到抗性构型的转变位于基因上游约4 kb处。此外，我们在克隆的DNA区域定位了两个重复序列。其中一个属于中等重复元件的CR1家族。它位于基因3'端约18 kb处，因此位于包含GAPDH的DNase I敏感区域之外。另一个重复元件属于一个未表征的家族。它位于基因上游，似乎处于从DNase I敏感状态到抗性状态的转变区域内。
